Pyridonyl guanidine F1F0-ATPase inhibitors and therapeutic uses thereof

ABSTRACT

The invention provides pyridonyl guanidine compounds that inhibit F 1 F 0 -ATPase, and methods of using pyridonyl guanidine compounds as therapeutic agents to treat medical disorders, such as an immune disorder, inflammatory condition, or cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national stage of International (PCT) PatentApplication Serial No. PCT/US2011/063945, filed Dec. 8, 2011, whichclaims the benefit of and priority to U.S. Provisional PatentApplication Ser. No. 61/420,934, filed Dec. 8, 2010, the contents ofeach application are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention provides inhibitors of F₁F₀-ATPases (e.g., mitochondrialF₁F₀-ATPases) and their therapeutic use. In particular, the inventionprovides pyridonyl guanidine compounds that inhibit F₁F₀-ATPase, andmethods of using pyridonyl guanidine compounds as therapeutic agents totreat a number of medical conditions.

BACKGROUND

Multicellular organisms exert precise control over cell number. Abalance between cell proliferation and cell death achieves thishomeostasis. Cell death occurs in nearly every type of vertebrate cellvia necrosis or through a suicidal form of cell death, known asapoptosis. Apoptosis is triggered by a variety of extracellular andintracellular signals that engage a common, genetically programmed deathmechanism.

Multicellular organisms use apoptosis to instruct damaged or unnecessarycells to destroy themselves for the good of the organism. Control of theapoptotic process therefore is very important to normal development, forexample, fetal development of fingers and toes requires the controlledremoval, by apoptosis, of excess interconnecting tissues, as does theformation of neural synapses within the brain. Similarly, controlledapoptosis is responsible for the sloughing off of the inner lining ofthe uterus (the endometrium) at the start of menstruation. Whileapoptosis plays an important role in tissue sculpting and normalcellular maintenance, it is also a component of the primary defenseagainst cells and invaders (e.g., viruses) which threaten the well beingof the organism.

Not surprisingly many diseases are associated with dysregulation ofapoptotic cell death. Experimental models have established acause-effect relationship between aberrant apoptotic regulation and thepathagenicity of various neoplastic, autoimmune and viral diseases. Forinstance, in the cell-mediated immune response, effector cells (e.g.,cytotoxic T lymphocytes “CTLs”) destroy virus-infected cells by inducingthe infected cells to undergo apoptosis. The organism subsequentlyrelies on the apoptotic process to destroy the effector cells when theyare no longer needed. Autoimmunity is normally prevented by the CTLsinducing apoptosis in each other and even in themselves. Defects in thisprocess are associated with a variety of immune diseases such as lupuserythematosus and rheumatoid arthritis.

Multicellular organisms also use apoptosis to instruct cells withdamaged nucleic acids (e.g., DNA) to destroy themselves prior tobecoming cancerous. Some cancer-causing viruses overcome this safeguardby reprogramming infected (transformed) cells to abort the normalapoptotic process. For example, several human papilloma viruses (HPVs)have been implicated in causing cervical cancer by suppressing theapoptotic removal of transformed cells by producing a protein (E6) whichinactivates the p53 apoptosis promoter. Similarly, the Epstein-Barrvirus (EBV), the causative agent of mononucleosis and Burkitt'slymphoma, reprograms infected cells to produce proteins that preventnormal apoptotic removal of the aberrant cells thus allowing thecancerous cells to proliferate and to spread throughout the organism.

Still other viruses destructively manipulate a cell's apoptoticmachinery without directly resulting in the development of a cancer. Forexample, destruction of the immune system in individuals infected withthe human immunodeficiency virus (HIV) is thought to progress throughinfected CD4⁺ T cells (about 1 in 100,000) instructing uninfected sistercells to undergo apoptosis.

Some cancers that arise by non-viral means have also developedmechanisms to escape destruction by apoptosis. Melanoma cells, forinstance, avoid apoptosis by inhibiting the expression of the geneencoding Apaf-1. Other cancer cells, especially lung and colon cancercells, secrete high levels of soluble decoy molecules that inhibit theinitiation of CTL mediated clearance of aberrant cells. Faultyregulation of the apoptotic machinery has also been implicated invarious degenerative conditions and vascular diseases.

Controlled regulation of the apoptotic process and its cellularmachinery is important to the survival of multicellular organisms.Typically, the biochemical changes that occur in a cell instructed toundergo apoptosis occur in an orderly procession. However, as shownabove, flawed regulation of apoptosis can cause serious deleteriouseffects in the organism.

The need exists for improved compositions and methods for regulating theapoptotic processes in subjects afflicted with diseases and conditionscharacterized by faulty regulation of these processes (e.g., viralinfections, hyperproliferative autoimmune disorders, chronicinflammatory conditions, and cancers). The present invention addressesthis need and provides other related advantages.

SUMMARY

The invention provides pyridonyl guanidine compounds that inhibitF₁F₀-ATPase (e.g., mitochondrial F₁F₀-ATPase), pharmaceuticalcompositions comprising pyridonyl guanidine compounds, and methods ofusing such compounds and pharmaceutical compositions to treat a numberof medical conditions. Accordingly, one aspect of the invention providesa family of compounds represented by Formula I:

including all stereoisomers, geometric isomers, and tautomers; or apharmaceutically acceptable salt or solvate of any of the foregoing,wherein the variables are as defined in the detailed description. Theforegoing compounds can be present in a pharmaceutical compositioncomprising a compound described herein and a pharmaceutically acceptablecarrier.

Another aspect of the invention provides a method of treating a subjectsuffering from a medical disorder. The method comprises administering tothe subject a therapeutically effective amount of one or more pyridonylguanidine compounds described herein, e.g., a compound of Formula I, inorder to ameliorate a symptom of the disorder. A large number ofdisorders can be treated using the pyridonyl guanidine compoundsdescribed herein. For example, the compounds described herein can beused to treat an immune disorder or inflammatory disorder, such asrheumatoid arthritis, psoriasis, chronic graft-versus-host disease,acute graft-versus-host disease, Crohn's disease, inflammatory boweldisease, multiple sclerosis, systemic lupus erythematosus, Celiac Sprue,idiopathic thrombocytopenic thrombotic purpura, myasthenia gravis,Sjogren's syndrome, scleroderma, ulcerative colitis, asthma, epidermalhyperplasia, and other medical disorders described herein. The compoundsdescribed herein can also be used to treat a cardiovascular disease,myeloma, lymphoma, cancer, or bacterial infection.

Another aspect of the invention provides a method of inhibiting anF₁F₀-ATPase, for example, a mitochondrial F₁F₀-ATPase. The methodcomprises exposing the F₁F₀-ATPase to a compound described herein, suchas a compound of Formula I, to inhibit said F₁F₀-ATPase.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides pyridonyl guanidine compounds that inhibitF₁F₀-ATPase (e.g., mitochondrial F₁F₀-ATPase), pharmaceuticalcompositions comprising the pyridonyl guanidine compounds, and methodsof using the pyridonyl guanidine compounds and pharmaceuticalcompositions in therapy.

Exemplary compositions and methods of the present invention aredescribed in more detail in the following sections: I. Modulators ofF₁F₀-ATPase Activity; II. Pyridonyl Guanidine Compounds; III.Therapeutic Applications of Pyridonyl Guanidine Compounds, and IV.Pharmaceutical Compositions, Formulations, and Exemplary AdministrationRoutes and Dosing Considerations. Aspects of the invention described inone particular section are not to be limited to any particular section.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as“Comprehensive Organic Synthesis” (B. M. Trost & I. Fleming, eds.,1991-1992); “Molecular cloning: a laboratory manual” Second Edition(Sambrook et al., 1989); “Oligonucleotide synthesis” (M. J. Gait, ed.,1984); “Animal cell culture” (R. I. Freshney, ed., 1987); the series“Methods in enzymology” (Academic Press, Inc.); “Handbook ofexperimental immunology” (D. M. Weir & C. C. Blackwell, eds.); “Genetransfer vectors for mammalian cells” (J. M. Miller & M. P. Calos, eds.,1987); “Current protocols in molecular biology” (F. M. Ausubel et al.,eds., 1987, and periodic updates); “PCR: the polymerase chain reaction”(Mullis et al., eds., 1994); and “Current protocols in immunology” (J.E. Coligan et al., eds., 1991), each of which is herein incorporated byreference in its entirety.

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, the term “guanidine” refers to a compound having thefollowing core structure:

including pharmaceutically acceptable salt forms.

The term “alkyl” refers to a saturated straight or branched hydrocarbon,such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms,referred to herein as C₁-C₁₂alkyl, C₁-C₁₀alkyl, and C₁-C₆alkyl,respectively. Exemplary alkyl groups include, but are not limited to,methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl,2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl,2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl,4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl,2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,hexyl, heptyl, octyl, etc.

The term “haloalkyl” refers to an alkyl group that is substituted withat least one halogen. For example, —CH₂F, —CHF₂, —CF₃, —CH₂CF₃, —CF₂CF₃,and the like.

The term “hydroxyalkyl” refers to an alkyl group that is substitutedwith a hydroxyl group.

The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic,or bridged cyclic (e.g., adamantyl)hydrocarbon group of 3-12, 3-8, 4-8,or 4-6 carbons, referred to herein, e.g., as “C₄₋₈cycloalkyl,” derivedfrom a cycloalkane. Exemplary cycloalkyl groups include cyclohexyl,cyclopentyl, cyclobutyl, and cyclopropyl.

The term “cycloalkylene” refers to a divalent (i.e., diradical)saturated cyclic, bicyclic, or bridged cyclic (e.g.,adamantyl)hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referredto herein, e.g., as “C₄₋₈cycloalkylene,” derived from a cycloalkane.Unless specified otherwise, the cycloalkylene may be substituted at oneor more ring positions with, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, amino, amido, carboxylic acid, —C(O)alkyl, —CO₂alkyl,carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide,ketone, aldehyde, ester, heterocyclyl, aryl, heteroaryl, —CF₃, —CN, orthe like. In certain embodiments, the cycloalkylene group is substitutedwith 1, 2, or 3 substituents independently selected from the groupconsisting of halogen, hydroxyl, alkoxyl, and amino. In certain otherembodiments, the cycloalkylene group is not substituted, i.e., it isunsubstituted. Exemplary cycloalkylene groups include

The term “aralkyl” refers to an alkyl group substituted with an arylgroup.

The term “heteroaralkyl” refers to an alkyl group substituted with aheteroaryl group.

The term “alkenyl” refers to an unsaturated straight or branchedhydrocarbon having at least one carbon-carbon double bond, such as astraight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referredto herein as C₂₋C₁₂alkenyl, C₂₋C₁₀alkenyl, and C₂₋C₆alkenyl,respectively. Exemplary alkenyl groups include, but are not limited to,vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl,hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl,4-(2-methyl-3-butene)-pentenyl, etc.

The term “alkynyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon triple bond, suchas a straight or branched group of 2-12, 2-8, or 2-6 carbon atoms,referred to herein as C₂-C₁₂alkynyl, C₂₋C₈alkynyl, and C₂₋C₆alkynyl,respectively. Exemplary alkynyl groups include, but are not limited to,ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl,4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl, etc.

The term “aryl” is art-recognized and refers to a carbocyclic aromaticgroup. Representative aryl groups include phenyl, naphthyl, anthracenyl,and the like. Unless specified otherwise, the aromatic ring may besubstituted at one or more ring positions with, for example, halogen,azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,amino, nitro, sulfhydryl, amino, amido, carboxylic acid, —C(O)alkyl,—CO₂alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido,sulfonamide, ketone, aldehyde, ester, heterocyclyl, heteroaryl, —CF₃,—CN, or the like. The term “aryl” also includes polycyclic ring systemshaving two or more carbocyclic rings in which two or more carbons arecommon to two adjoining rings (the rings are “fused rings”) wherein atleast one of the rings is aromatic, and the other ring(s) may be, forexample, cycloalkyl, cycloalkenyl, cycloalkynyl, and/or aryl. The term“haloaryl” refers to an aryl group that is substituted with at least onehalogen. In certain embodiments, the aromatic group is not substituted,i.e., it is unsubstituted.

The term “phenylene” refers to a multivalent radical (e.g., a divalentor trivalent radical) of benzene. To illustrate, a divalent valentradical of benzene is illustrated by the formula

The terms “heterocyclyl” or “heterocyclic group” are art-recognized andrefer to saturated, partially unsaturated, or aromatic 3- to 10-memberedring structures, alternatively 3- to 7-membered rings, whose ringstructures include one to four heteroatoms, such as nitrogen, oxygen,and sulfur. Heterocycles may also be mono-, bi-, or other multi-cyclicring systems. A heterocycle may be fused to one or more aryl, partiallyunsaturated, or saturated rings. Heterocyclyl groups include, forexample, biotinyl, chromenyl, dihydrofuryl, dihydroindolyl,dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl,imidazolidinyl, isoquinolyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, oxolanyl, oxazolidinyl, phenoxanthenyl, piperazinyl,piperidinyl, pyranyl, pyrazolidinyl, pyrazolinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, tetrahydrofuryl,tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl,thiazolidinyl, thiolanyl, thiomorpholinyl, thiopyranyl, xanthenyl,lactones, lactams such as azetidinones and pyrrolidinones, sultams,sultones, and the like. Unless specified otherwise, the heterocyclicring is optionally substituted at one or more positions withsubstituents such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido,amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy,cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl,heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato,phosphinato, sulfate, sulfide, sulfonamido, sulfonyl and thiocarbonyl.In certain embodiments, the heterocyclcyl group is not substituted,i.e., it is unsubstituted.

The term “heteroaryl” is art-recognized and refers to aromatic groupsthat include at least one ring heteroatom. In certain instances, aheteroaryl group contains 1, 2, 3, or 4 ring heteroatoms. Representativeexamples of heteroaryl groups includes pyrrolyl, furanyl, thiophenyl,imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl,pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Unless specifiedotherwise, the heteroaryl ring may be substituted at one or more ringpositions with, for example, halogen, azide, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,amido, carboxylic acid, —C(O)alkyl, —CO₂alkyl, carbonyl, carboxyl,alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester,heterocyclyl, aryl, —CF₃, —CN, or the like. The term “heteroaryl” alsoincludes polycyclic ring systems having two or more rings in which twoor more carbons are common to two adjoining rings (the rings are “fusedrings”) wherein at least one of the rings is heteroaromatic, and theother ring(s) may be, for example, cycloalkyl, cycloalkenyl,cycloalkynyl, and/or aryl.

The term “heteroarylene” refers to a multi-valent (e.g., di-valent ortrivalent) aromatic group that comprises at least one ring heteroatom.An exemplary “heteroarylene” is pyridinylene, which is a multi-valentradical of pyridine. For example, a divalent radical of pyridine isillustrated by the formula

The terms ortho, meta and para are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formula:

wherein R⁵⁰ and R⁵¹ each independently represent hydrogen, alkyl,alkenyl, or —(CH₂)_(m)—R⁶¹; or R⁵⁰ and R⁵¹, taken together with the Natom to which they are attached complete a heterocycle having from 4 to8 atoms in the ring structure; wherein R⁶¹ is aryl, cycloalkyl,cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In certain embodiments, R⁵⁰ and R⁵¹ eachindependently represent hydrogen or alkyl.

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as may berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R⁶¹, where m and R⁶¹ are described above.

The term “amide” or “amido” as used herein refers to a radical of theform —R_(a)C(O)N(R_(b))—, —R_(a)C(O)N(R_(b))R_(c)—, —C(O)NR_(b)R_(c), or—C(O)NH₂, wherein R_(a), R_(b) and R_(c) are each independently selectedfrom alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl,carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl,heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, and nitro. Theamide can be attached to another group through the carbon, the nitrogen,R_(b), R_(c), or R_(a). The amide also may be cyclic, for example R_(b)and R_(c), R_(a) and R_(b), or R_(a) and R_(c) may be joined to form a3- to 12-membered ring, such as a 3- to 10-membered ring or a 5- to6-membered ring. The term “carboxamido” refers to the structure—C(O)NR_(b)R_(c).

The term “sulfonamide” or “sulfonamido” as used herein refers to aradical having the structure —N(R_(r))—S(O)₂—R_(s)— or—S(O)₂—N(R_(r))R_(s), where R_(r), and R_(s) can be, for example,hydrogen, alkyl, aryl, cycloalkyl, and heterocyclyl. Exemplarysulfonamides include alkylsulfonamides (e.g., where R_(s) is alkyl),arylsulfonamides (e.g., where R_(s) is aryl), cycloalkyl sulfonamides(e.g., where R_(s) is cycloalkyl), and heterocyclyl sulfonamides (e.g.,where R_(s) is heterocyclyl), etc.

The term “sulfonyl” as used herein refers to a radical having thestructure R_(u)SO₂—, where R_(u) can be alkyl, aryl, cycloalkyl, andheterocyclyl, e.g., alkylsulfonyl. The term “alkylsulfonyl” as usedherein refers to an alkyl group attached to a sulfonyl group.

The symbol “

” indicates a point of attachment.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asgeometric isomers, enantiomers or diastereomers. The term“stereoisomers” when used herein consist of all geometric isomers,enantiomers or diastereomers. These compounds may be designated by thesymbols “R” or “S,” depending on the configuration of substituentsaround the stereogenic carbon atom. Stereoisomers include enantiomersand diastereomers. Mixtures of enantiomers or diastereomers may bedesignated “(±)” in nomenclature, but the skilled artisan will recognizethat a structure may denote a chiral center implicitly. Unless indicatedotherwise, generic chemical structures and graphical representations ofspecific compounds encompass all stereoisomers.

Individual stereoisomers of compounds of the present invention can beprepared synthetically from commercially available starting materialsthat contain asymmetric or stereogenic centers, or by preparation ofracemic mixtures followed by resolution methods well known to those ofordinary skill in the art. These methods of resolution are exemplifiedby (1) attachment of a mixture of enantiomers to a chiral auxiliary,separation of the resulting mixture of diastereomers byrecrystallization or chromatography and liberation of the optically pureproduct from the auxiliary, (2) salt formation employing an opticallyactive resolving agent, or (3) direct separation of the mixture ofoptical enantiomers on chiral chromatographic columns. Stereoisomericmixtures can also be resolved into their component stereoisomers by wellknown methods, such as chiral-phase gas chromatography, chiral-phasehigh performance liquid chromatography, crystallizing the compound as achiral salt complex, or crystallizing the compound in a chiral solvent.Stereoisomers can also be obtained from stereomerically-pureintermediates, reagents, and catalysts by well known asymmetricsynthetic methods.

Geometric isomers can also exist in the compounds of the presentinvention. The symbol

denotes a bond that may be a single, double or triple bond as describedherein. The present invention encompasses the various geometric isomersand mixtures thereof resulting from the arrangement of substituentsaround a carbon-carbon double bond or arrangement of substituents arounda carbocyclic ring. Substituents around a carbon-carbon double bond aredesignated as being in the “Z” or “E” configuration wherein the terms“Z” and “E” are used in accordance with IUPAC standards. Unlessotherwise specified, structures depicting double bonds encompass boththe “E” and “Z” isomers.

Substituents around a carbon-carbon double bond alternatively can bereferred to as “cis” or “trans,” where “cis” represents substituents onthe same side of the double bond and “trans” represents substituents onopposite sides of the double bond. The arrangement of substituentsaround a carbocyclic ring are designated as “cis” or “trans.” The term“cis” represents substituents on the same side of the plane of the ringand the term “trans” represents substituents on opposite sides of theplane of the ring. Mixtures of compounds wherein the substituents aredisposed on both the same and opposite sides of plane of the ring aredesignated “cis/trans.”

Certain compounds described herein may exist as a single tautomer or asa mixture of tautomers. For example, certain guanidine compounds havinga hydrogen atom attached to at least one of the guanidine nitrogen atomscan exist as a single tautomer or a mixture of tautomers. To illustrate,depending upon the substituents attached at the R¹, R² and R³ positions,the guanidine compound may exist as a single tautomer represented by A,B, or C, or as mixture of two or more of A, B, and C.

The compounds disclosed herein can exist in solvated as well asunsolvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms.

The invention also embraces isotopically labeled compounds of theinvention which are identical to those recited herein, except that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O,¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively.

Certain isotopically-labeled disclosed compounds (e.g., those labeledwith ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C)isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Isotopically labeled compounds of the invention cangenerally be prepared by following procedures analogous to thosedisclosed in the e.g., Examples herein by substituting an isotopicallylabeled reagent for a non-isotopically labeled reagent.

The term “IC₅₀” is art-recognized and refers to the concentration of acompound that is required for 50% inhibition of its target.

The term “EC₅₀” is art-recognized and refers to the concentration of acompound at which 50% of its maximal effect is observed.

The terms “subject” and “patient” refer to organisms to be treated bythe methods of the present invention. Such organisms preferably include,but are not limited to, mammals (e.g., murines, simians, equines,bovines, porcines, canines, felines, and the like), and most preferablyincludes humans. In the context of the invention, the terms “subject”and “patient” generally refer to an individual who will receive or whohas received treatment (e.g., administration of a compound of thepresent invention and optionally one or more other agents) for acondition characterized by the dysregulation of apoptotic processes.

As used herein, the term “effective amount” refers to the amount of acompound sufficient to effect beneficial or desired results. Aneffective amount can be administered in one or more administrations,applications or dosages and is not intended to be limited to aparticular formulation or administration route. As used herein, the term“treating” includes any effect, e.g., lessening, reducing, modulating,ameliorating or eliminating, that results in the improvement of thecondition, disease, disorder, and the like, or ameliorating a symptomthereof.

The phrase “pathologically proliferating or growing cells” refers to alocalized population of proliferating cells in an animal that is notgoverned by the usual limitations of normal growth.

As used herein, the term “un-activated target cell” refers to a cellthat is either in the G_(o) phase or one to which a stimulus has notbeen applied.

As used herein, the term “activated target lymphoid cell” refers to alymphoid cell that has been primed with an appropriate stimulus to causea signal transduction cascade, or alternatively, a lymphoid cell that isnot in G_(o) phase. Activated lymphoid cells may proliferate, undergoactivation induced cell death, or produce one or more cytotoxins,cytokines, or other related membrane-associated proteins characteristicof the cell type (e.g., CD8⁺ or CD4⁺). They are also capable ofrecognizing and binding any target cell that displays a particularantigen on its surface, and subsequently releasing its effectormolecules.

As used herein, the term “activated cancer cell” refers to a cancer cellthat has been primed with an appropriate stimulus to cause signaltransduction. An activated cancer cell may or may not be in the G_(o)phase.

An activating agent is a stimulus that upon interaction with a targetcell results in a signal transduction cascade. Examples of activatingstimuli include, but are not limited to, small molecules, radiantenergy, and molecules that bind to cell activation cell surfacereceptors. Responses induced by activation stimuli can be characterizedby changes in, among others, intracellular Ca²⁺, superoxide, or hydroxylradical levels; the activity of enzymes like kinases or phosphatases; orthe energy state of the cell. For cancer cells, activating agents alsoinclude transforming oncogenes.

As used herein, the term “dysregulation of the process of cell death”refers to any aberration in the ability (e.g., predisposition) of a cellto undergo cell death via either necrosis or apoptosis. Dysregulation ofcell death is associated with or induced by a variety of conditions,including for example, immune disorders (e.g., systemic lupuserythematosus, autoimmune disorders, rheumatoid arthritis,graft-versus-host disease, myasthenia gravis, Sjogren's syndrome, etc.),chronic inflammatory conditions (e.g., psoriasis, asthma and Crohn'sdisease), hyperproliferative disorders (e.g., tumors, B cell lymphomas,T cell lymphomas, etc.), viral infections (e.g., herpes, papilloma,HIV), and other conditions such as osteoarthritis and atherosclerosis.

It should be noted that when the dysregulation is induced by orassociated with a viral infection, the viral infection may or may not bedetectable at the time dysregulation occurs or is observed. That is,viral-induced dysregulation can occur even after the disappearance ofsymptoms of viral infection.

A “hyperproliferative disorder,” as used herein refers to any conditionin which a localized population of proliferating cells in an animal isnot governed by the usual limitations of normal growth. Examples ofhyperproliferative disorders include tumors, neoplasms, lymphomas andthe like. A neoplasm is said to be benign if it does not undergoinvasion or metastasis and malignant if it does either of these. Ametastatic cell or tissue means that the cell can invade and destroyneighboring body structures. Hyperplasia is a form of cell proliferationinvolving an increase in cell number in a tissue or organ, withoutsignificant alteration in structure or function. Metaplasia is a form ofcontrolled cell growth in which one type of fully differentiated cellsubstitutes for another type of differentiated cell. Metaplasia canoccur in epithelial or connective tissue cells. A typical metaplasiainvolves a somewhat disorderly metaplastic epithelium.

The pathological growth of activated lymphoid cells often results in animmune disorder or a chronic inflammatory condition. As used herein, theterm “immune disorder” refers to any condition in which an organismproduces antibodies or immune cells which recognize the organism's ownmolecules, cells or tissues. Non-limiting examples of immune disordersinclude autoimmune disorders, immune hemolytic anemia, immune hepatitis,Berger's disease or IgA nephropathy, Celiac Sprue, chronic fatiguesyndrome, Crohn's disease, dermatomyositis, fibromyalgia,graft-versus-host disease, Grave's disease, Hashimoto's thyroiditis,idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis,myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,scleroderma, Sjorgren syndrome, systemic lupus erythematosus, type 1diabetes, ulcerative colitis, vitiligo, tuberculosis, and the like.

As used herein, the term “chronic inflammatory condition” refers to acondition wherein the organism's immune cells are activated. Such acondition is characterized by a persistent inflammatory response withpathologic sequelae. This state is characterized by infiltration ofmononuclear cells, proliferation of fibroblasts and small blood vessels,increased connective tissue, and tissue destruction. Examples of chronicinflammatory diseases include, but are not limited to, Crohn's disease,psoriasis, chronic obstructive pulmonary disease, inflammatory boweldisease, multiple sclerosis, and asthma. Immune diseases such asrheumatoid arthritis and systemic lupus erythematosus can also result ina chronic inflammatory state.

As used herein, the term “co-administration” refers to theadministration of at least two agent(s) (e.g., a compound of the presentinvention) or therapies to a subject. In some embodiments, theco-administration of two or more agents/therapies is concurrent. Inother embodiments, a first agent/therapy is administered prior to asecond agent/therapy. Those of skill in the art understand that theformulations and/or routes of administration of the variousagents/therapies used may vary. The appropriate dosage forco-administration can be readily determined by one skilled in the art.In some embodiments, when agents/therapies are co-administered, therespective agents/therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents/therapies lowers the requisite dosage of a known potentiallyharmful (e.g., toxic) agent(s).

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with a carrier, inert or active, makingthe composition especially suitable for diagnostic or therapeutic use invivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions (e.g., such as an oil/wateror water/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. [1975]).

As used herein, the term “pharmaceutically acceptable salt” refers toany pharmaceutically acceptable salt (e.g., acid or base) of a compoundof the present invention which, upon administration to a subject, iscapable of providing a compound of this invention or an activemetabolite or residue thereof. As is known to those of skill in the art,“salts” of the compounds of the present invention may be derived frominorganic or organic acids and bases. Examples of acids include, but arenot limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,benzenesulfonic acid, and the like. Other acids, such as oxalic, whilenot in themselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts.

Examples of bases include, but are not limited to, alkali metals (e.g.,sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound.

As used herein, the term “modulate” refers to the activity of a compound(e.g., a compound of the present invention) to affect (e.g., to promoteor retard) an aspect of cellular function, including, but not limitedto, cell growth, proliferation, apoptosis, and the like.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes andmethods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions ofthe present invention that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the present invention that consist essentially of, or consist of, therecited processing steps.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified. Further, if a variable is not accompanied bya definition, then the previous definition of the variable controls.

I. Modulators of F₁F₀-ATPase Activity

In some embodiments, the present invention regulates F₁F₀-ATPaseactivity (e.g., mitochondrial F₁F₀-ATPase activity) through the exposureof cells to compounds of the present invention. In some embodiments, thecompounds inhibit ATP synthesis and ATP hydrolysis. The effect of thecompounds can be measured by detecting any number of cellular changes.For example, mitochondrial F₁F₀-ATPase activity and/or cell death may beassayed as described herein and in the art. In some embodiments, celllines are maintained under appropriate cell culturing conditions (e.g.,gas (CO₂), temperature and media) for an appropriate period of time toattain exponential proliferation without density dependent constraints.Cell number and or viability are measured using standard techniques,such as trypan blue exclusion/hemo-cytometry, or an Alamar Blue or MTTdye conversion assay. Alternatively, the cell may be analyzed for theexpression of genes or gene products associated with aberrations inapoptosis or necrosis.

In some embodiments, exposing the compounds of the present invention toa cell induces apoptosis. In some embodiments, the present inventioninduces apoptosis or arrest of cell proliferation through interactingwith the mitochondrial F₁F₀-ATPase. In some embodiments, the compoundsof the present invention inhibit mitochondrial F₁F₀-ATPase activitythrough binding the OSCP. In some embodiments, the compounds of thepresent invention bind the junction between the OSCP and the F₁ subunitof the mitochondrial F₁F₀-ATPase. In some embodiments, the compounds ofthe present invention bind the F₁ subunit. In certain embodiments,screening assays of the present invention permit detection of bindingpartners of the OSCP, F₁, or OSCP/F₁ junction.

In some embodiments, exposing a compound of the present invention to acell induces apoptosis. In some embodiments, the present inventioncauses an initial increase in cellular ROS levels (e.g., O₂ ⁻). Infurther embodiments, exposure of the compounds of the present inventionto a cell causes an increase in cellular O₂ ⁻ levels. In still furtherembodiments, the increase in cellular O₂ ⁻ levels resulting from thecompounds of the present invention is detectable with a redox-sensitiveagent that reacts specifically with O₂ ⁻ (e.g., dihydroethidium (DHE)).

In some embodiments, the present invention causes a collapse of a cell'smitochondrial transmembrane potential (ΔΨ_(m)). In some embodiments, acollapse of a cell's mitochondrial ΔΨ_(m), resulting from the presentinvention is detectable with a mitochondria-selective potentiometricprobe (e.g., 3,3′-Dihexyloxacarbocyanine iodide, DiOC₆). In furtherembodiments, a collapse of a cell's mitochondrial ΔΨ_(m) resulting fromthe present invention occurs after an initial increase in cellular O₂ ⁻levels.

In some embodiments, the present invention enables caspase activation.In other embodiments, the present invention causes the release ofcytochrome c from mitochondria. In further embodiments, the presentinvention alters cystolic cytochrome c levels. In still otherembodiments, altered cystolic cytochrome c levels resulting from thepresent invention are detectable by immunoblotting cytosolic fractions.In some embodiments, diminished cystolic cytochrome c levels resultingfrom the present invention are detectable after a period of time (e.g.,10 hours). In further preferred embodiments, diminished cystoliccytochrome c levels resulting from the present invention are detectableafter 5 hours.

In other embodiments, the present invention causes the opening of themitochondrial permeability transition pore. In some embodiments, thecellular release of cytochrome c resulting from the present invention isconsistent with a collapse of mitochondrial ΔΨ_(m). In still furtherpreferred embodiments, the present invention causes an increase incellular O₂ ⁻ levels after a mitochondrial ΔΨ_(m) collapse and a releaseof cytochrome c. In further preferred embodiments, a rise in cellular O₂⁻ levels is caused by a mitochondrial ΔΨ_(m) collapse and release ofcytochrome c resulting from the present invention.

In other embodiments, the present invention causes cellular caspaseactivation. In some embodiments, caspase activation resulting from thepresent invention is measurable with a pan-caspase sensitive fluorescentsubstrate (e.g., FAM-VAD-fmk). In still further embodiments, caspaseactivation resulting from the present invention tracks with a collapseof mitochondrial ΔΨ_(m). In other embodiments, the present inventioncauses an appearance of hypodiploid DNA. In some embodiments, anappearance of hypodiploid DNA resulting from the present invention isslightly delayed with respect to caspase activation.

In some embodiments, the molecular target for the present invention isfound within mitochondria. In further embodiments, the molecular targetof the present invention involves the mitochondrial ATPase. The primarysources of cellular ROS include redox enzymes and the mitochondrialrespiratory chain (hereinafter MRC). In some embodiments, cytochrome coxidase (complex IV of the MRC) inhibitors (e.g., NaN₃) preclude apresent invention dependent increase in cellular ROS levels. In otherpreferred embodiments, the ubiquinol-cytochrome c reductase component ofMRC complex III inhibitors (e.g., FK506) preclude a present inventiondependent increase in ROS levels.

II. Pyridonyl Guanidine Compounds

One aspect of the invention provides a family of compounds representedby Formula I:

including all stereoisomers, geometric isomers, and tautomers; or apharmaceutically acceptable salt or solvate of any of the foregoing;wherein:

A¹ is phenylene or a six-membered heteroarylene;

A² is

X is halogen, haloalkyl, C₁-C₆alkoxy, —N(H)(R⁸), or —OP(O)(OR¹¹)₂;

R¹ represents independently for each occurrence halogen, alkyl,haloalkyl, hydroxyl, C₁-C₆alkoxy, or cyano;

R² is hydrogen or alkyl;

R³ is aryl, aralkyl, cycloalkyl, —(C(R⁶)₂)_(m)-cycloalkyl, heteroaryl,heteroaralkyl, heterocycloalkyl, —(C(R⁶)₂)_(m)-heterocycloalkyl, alkyl,haloalkyl, hydroxylalkyl, —(C(R⁶)₂)_(m)-alkoxyl, or —(C(R⁸)₂)_(m)—CN,wherein said aryl, aralkyl, cycloalkyl, heteroaryl, heteroaralkyl, andheterocycloalkyl are each optionally substituted with 1, 2, or 3substituents independently selected from the group consisting ofhalogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, C₁-C₆alkoxy, and cyano;

R⁴ is hydrogen, alkyl, or —C(O)R⁷; or R³ and R⁴ are taken together withthe nitrogen atom to which they are attached to form a 3 to 7 memberedheterocyclic ring optionally substituted with 1, 2, or 3 substituentsindependently selected from the group consisting of halogen, haloalkyl,hydroxyl, alkyl, cycloalkyl, and C₁-C₆alkoxy;

R⁵ is hydrogen, halogen, alkyl, alkoxyl, or —C(O)R⁷;

R⁶ represents independently for each occurrence hydrogen, alkyl, orcycloalkyl;

R⁷ represents independently for each occurrence alkyl or cycloalkyl;

R⁸ is hydrogen or alkyl;

R⁹ is alkyl, cycloalkyl, haloalkyl, —(C(R⁶)₂)_(m)-cycloalkyl,—(C(R⁶)₂)_(m)—CN, aryl, aralkyl, heteroaryl, or heteroaralkyl;

R¹⁰ is alkyl, cycloalkyl, —(C(R⁶)₂)_(m)-cycloalkyl, haloalkyl, orC₁-C₆alkoxy;

R¹¹ represents independently for each occurrence hydrogen or an alkalimetal;

n is 0, 1, 2, or 3; and

m is 1, 2, 3, 4, or 5.

Definitions of the variables in Formula I above encompass multiplechemical groups. The application contemplates embodiments where, forexample, i) the definition of a variable is a single chemical groupselected from those chemical groups set forth above, ii) the definitionis a collection of two or more of the chemical groups selected fromthose set forth above, and iii) the compound is defined by a combinationof variables in which the variables are defined by (i) or (ii), e.g.,such as where A¹ is phenylene, R¹ is halogen or haloalkyl, R² ishydrogen, and R⁵ is hydrogen.

Accordingly, in certain embodiments, A¹ is phenylene. In certainembodiments, A¹ is a six-membered heteroarylene, such as pyridinylene orpyrimidinylene. In certain embodiments, A¹ is pyridinylene.

In certain embodiments, A² is

In certain other embodiments, A² is

In certain other embodiments, A² is

In certain other embodiments,

In certain other embodiments, A² is

In certain other embodiments, A² is

In certain other embodiments, A² is

In certain other embodiments, A² is

In certain embodiments, R¹ is halogen or haloalkyl. In certainembodiments, R¹ is chloro, fluoro, or trifluoromethyl.

In certain embodiments, R² is hydrogen. In certain embodiments, R² isalkyl, such as methyl or ethyl. In certain embodiments, R⁴ is hydrogen.In certain embodiments, R⁴ is alkyl, such as methyl or ethyl. In certainembodiments, R² and R⁴ are hydrogen. In certain embodiments, R⁴ is—C(O)R⁷. In certain embodiments, R² and R⁴ are hydrogen.

In certain embodiments, R³ is aryl or aralkyl, each of which isoptionally substituted with 1, 2, or 3 substituents independentlyselected from the group consisting of halogen, haloalkyl, alkyl, andcycloalkyl. In certain embodiments, R³ is phenyl optionally substitutedwith 1, 2, or 3 substituents independently selected from the groupconsisting of halogen, haloalkyl, alkyl, and cycloalkyl. In certainembodiments, R³ is phenyl substituted with 1, 2, or 3 substituentsindependently selected from the group consisting of chloro, fluoro,trifluoromethyl, cyclopropyl, and (C₁-C₄)alkyl. In certain embodiments,R³ is phenyl substituted with 1 or 2 substituents independently selectedfrom the group consisting of chloro, fluoro, and trifluoromethyl.

In certain embodiments, R³ is benzyl optionally substituted with 1, 2,or 3 substituents independently selected from the group consisting ofhalogen, haloalkyl, alkyl, and cycloalkyl. In certain embodiments, R³ isbenzyl substituted with 1 or 2 substituents independently selected fromthe group consisting of chloro, fluoro, and trifluoromethyl. In certainembodiments, R³ is benzyl substituted with 1, 2, or 3 substituentsindependently selected from the group consisting of chloro, fluoro,trifluoromethyl, cyclopropyl, and (C₁-C₄)alkyl.

In certain embodiments, R³ is alkyl, hydroxyalkyl, cycloalkyl, or—(C(R⁶)₂)_(m)-alkoxyl, wherein said cycloalkyl is optionally substitutedwith 1 or 2 substituents independently selected from the groupconsisting of halogen, haloalkyl, hydroxyl, and alkyl. In certain otherembodiments, R³ is alkyl, hydroxyalkyl, or cycloalkyl, wherein saidcycloalkyl is optionally substituted with 1 or 2 substituentsindependently selected from the group consisting of halogen, haloalkyl,hydroxyl, and alkyl. In certain other embodiments, R³ is alkyl orcycloalkyl, wherein said cycloalkyl is optionally substituted with 1 or2 substituents independently selected from the group consisting ofhalogen, haloalkyl, and alkyl. In certain embodiments, R³ is heteroarylor heteroaralkyl, wherein said heteroaryl and heteroaralkyl are eachoptionally substituted with 1, 2, or 3 substituents independentlyselected from the group consisting of halogen, haloalkyl, and alkyl. Incertain embodiments, R³ is haloalkyl.

In certain embodiments, R⁵ is hydrogen. In certain embodiments, R⁵ ishalogen or alkyl. In certain embodiments, R⁵ is alkyl, such as methyl orethyl.

In certain embodiments, R⁶ is hydrogen. In certain embodiments, R⁶represents independently for each occurrence hydrogen or alkyl.

In certain embodiments, R⁷ is alkyl, such as methyl or ethyl.

In certain embodiments, R⁸ is hydrogen.

In certain embodiments, R⁹ is alkyl (such as (C₁-C₄)alkyl), haloalkyl(such as —CH₂CF₃), —(C(R⁶)₂)_(m)—CN, aryl (such as phenyl optionallysubstituted with 1, 2, or 3 substituents independently selected fromalkyl, halogen, and cyano), or heteroaryl (such as pyridinyl,imidazolyl, or isoxazolyl, each of which is optionally substituted with1, 2, or 3 substituents independently selected from alkyl, halogen, andcyano).

In certain embodiments, R¹⁰ is alkyl (such as (C₁-C₄)alkyl), orC₁-C₆alkoxy (such as methoxy or ethoxy).

In certain embodiments, n is 1 or 2. In certain embodiments, n is 1. Incertain embodiments, n is 2. In certain embodiments, m is 1 or 2.

In certain embodiments, the compound is represented by Formula I-A:

including all stereoisomers, geometric isomers, tautomers, or apharmaceutically acceptable salt or solvate of any of the foregoing;wherein:

R¹ and R² each represent independently for each occurrence hydrogen,chloro, fluoro, or —CF₃; and

R³ is aryl, aralkyl, heteroaryl, or heteroaralkyl, wherein said aryl,aralkyl, heteroaryl, and heteroaralkyl are each optionally substitutedwith 1, 2, or 3 substituents independently selected from the groupconsisting of halogen, haloalkyl, hydroxyl, alkyl, C₁-C₆alkoxy, andcyano.

Definitions of the variables in Formula I-A above encompass multiplechemical groups. The application contemplates embodiments where, forexample, i) the definition of a variable is a single chemical groupselected from those chemical groups set forth above, ii) the definitionis a collection of two or more of the chemical groups selected fromthose set forth above, and iii) the compound is defined by a combinationof variables in which the variables are defined by (i) or (ii), e.g.,such as where R¹ is chloro or fluoro, and R³ is aryl optionallysubstituted with 1, 2, or 3 substituents independently selected from thegroup consisting of halogen and haloalkyl.

Accordingly, in certain embodiments, R¹ and R² are independently chloroor fluoro.

In certain embodiments, R³ is aryl or aralkyl, each of which isoptionally substituted with 1, 2, or 3 substituents independentlyselected from the group consisting of halogen, haloalkyl, and alkyl. Incertain embodiments, R³ is phenyl optionally substituted with 1, 2, or 3substituents independently selected from the group consisting ofhalogen, haloalkyl, and alkyl. In certain embodiments, R³ is phenylsubstituted with 1, 2, or 3 substituents independently selected from thegroup consisting of chloro, fluoro, trifluoromethyl, cyclopropyl, and(C₁-C₄)alkyl. In certain embodiments, R³ is phenyl substituted with 1 or2 substituents independently selected from the group consisting ofchloro, fluoro, and trifluoromethyl.

In certain embodiments, R³ is benzyl optionally substituted with 1, 2,or 3 substituents independently selected from the group consisting ofhalogen, haloalkyl, and alkyl. In certain embodiments, R³ is benzylsubstituted with 1 or 2 substituents independently selected from thegroup consisting of chloro, fluoro, and trifluoromethyl. In certainembodiments, R³ is benzyl substituted with 1, 2, or 3 substituentsindependently selected from the group consisting of chloro, fluoro,trifluoromethyl, cyclopropyl, and (C₁-C₄)alkyl.

In certain embodiments, R³ is alkyl or cycloalkyl, wherein saidcycloalkyl is optionally substituted with 1, 2, or 3 substituentsindependently selected from the group consisting of halogen, haloalkyl,and alkyl. In certain embodiments, R³ is heteroaryl or heteroaralkyl,wherein said heteroaryl and heteroaralkyl are each optionallysubstituted with 1, 2, or 3 substituents independently selected from thegroup consisting of halogen, haloalkyl, and alkyl. In certainembodiments, R³ is haloalkyl.

In certain embodiments, the compound is represented by Formula I-B:

or a pharmaceutically acceptable salt thereof; wherein:

R¹ and R² each represent independently for each occurrence hydrogen,chloro, fluoro, or —CF₃; and

R³ is aryl, aralkyl, heteroaryl, or heteroaralkyl, wherein said aryl,aralkyl, heteroaryl, and heteroaralkyl are each optionally substitutedwith 1, 2, or 3 substituents independently selected from the groupconsisting of halogen, haloalkyl, and alkyl.

Definitions of the variables in Formula I-B above encompass multiplechemical groups. The application contemplates embodiments where, forexample, i) the definition of a variable is a single chemical groupselected from those chemical groups set forth above, ii) the definitionis a collection of two or more of the chemical groups selected fromthose set forth above, and iii) the compound is defined by a combinationof variables in which the variables are defined by (i) or (ii), e.g.,such as where R¹ is chloro or fluoro, and R³ is aryl optionallysubstituted with 1, 2, or 3 substituents independently selected from thegroup consisting of halogen and haloalkyl.

Accordingly, in certain embodiments, R¹ and R² are independently chloroor fluoro.

In certain embodiments, R³ is aryl or aralkyl, each of which isoptionally substituted with 1, 2, or 3 substituents independentlyselected from the group consisting of halogen, haloalkyl, and alkyl. Incertain embodiments, R³ is phenyl optionally substituted with 1, 2, or 3substituents independently selected from the group consisting ofhalogen, haloalkyl, and alkyl. In certain embodiments, R³ is phenylsubstituted with 1, 2, or 3 substituents independently selected from thegroup consisting of chloro, fluoro, trifluoromethyl, cyclopropyl, and(C₁-C₄)alkyl. In certain embodiments, R³ is phenyl substituted with 1 or2 substituents independently selected from the group consisting ofchloro, fluoro, and trifluoromethyl.

In certain embodiments, R³ is benzyl optionally substituted with 1, 2,or 3 substituents independently selected from the group consisting ofhalogen, haloalkyl, and alkyl. In certain embodiments, R³ is benzylsubstituted with 1 or 2 substituents independently selected from thegroup consisting of chloro, fluoro, and trifluoromethyl. In certainembodiments, R³ is benzyl substituted with 1, 2, or 3 substituentsindependently selected from the group consisting of chloro, fluoro,trifluoromethyl, cyclopropyl, and (C₁-C₄)alkyl.

In certain embodiments, R³ is alkyl or cycloalkyl, wherein saidcycloalkyl is optionally substituted with 1, 2, or 3 substituentsindependently selected from the group consisting of halogen, haloalkyl,and alkyl. In certain embodiments, R³ is heteroaryl or heteroaralkyl,wherein said heteroaryl and heteroaralkyl are each optionallysubstituted with 1, 2, or 3 substituents independently selected from thegroup consisting of halogen, haloalkyl, and alkyl. In certainembodiments, R³ is haloalkyl.

The description above describes multiple embodiments providingdefinitions for variables used herein. The application specificallycontemplates all combinations of such embodiments. For example, theapplication contemplates particular combinations of embodiments relatingto Formula I, such as where A¹ is phenylene, R¹ is halogen or haloalkyl,and n is 1. Further, for example, the application contemplatesparticular combinations of embodiments relating to Formula I-A, such aswhere R¹ and R² are independently chloro or fluoro, and R³ is phenylsubstituted with 1 or 2 substituents independently selected from thegroup consisting of chloro, fluoro, and trifluoromethyl.

In certain other embodiments, the compound is one of the compoundslisted in Table 1 below or a pharmaceutically acceptable salt thereof.It is understood that the foregoing compounds can be combined with apharmaceutically acceptable carrier to produce a pharmaceuticalcomposition.

TABLE 1

No. A X Y Z I-1  3-chlorophenyl

H 3-chlorophenyl I-2  4-chlorophenyl

H 4-chlorophenyl I-3  3-fluorophenyl

H 3,5-dichlorophenyl I-4  4-fluorophenyl

H 3-chloro-4-fluorophenyl I-5  3,4-dichlorophenyl

H 3-chloro-5-fluorophenyl I-6  3,4-difluorophenyl

H 3-trifluoromethylphenyl I-7  4-trifluoromethylphenyl

H 3-cyclopropylphenyl I-8  3-chlorophenyl

H 3-tert-butylphenyl I-9  4-chlorophenyl

H 2-cyclopropylphenyl I-10 3-fluorophenyl

H 2-cyclopropyl-4- fluorophenyl I-11 4-fluorophenyl

H 3-chlorobenzyl I-12 3,4-dichlorophenyl

H 4-chlorobenzyl I-13 3,4-difluorophenyl

H 3-fluorobenzyl I-14 4-trifluoromethylphenyl

H 4-fluorobenzyl I-15 3-chlorophenyl

H 3-chloro-5-fluorobenzyl I-16 4-chlorophenyl

H 3,5-dichlorobenzyl I-17 3-fluorophenyl

H 3,5-difluorobenzyl I-18 4-fluorophenyl

H 3-cyclopropylbenzyl I-19 3,4-dichlorophenyl

H 3-trifluoromethylbenzyl I-20 3,4-difluorophenyl

H 4-trifluoromethylbenzyl I-21 4-trifluoromethylphenyl

H cyclopropyl I-22 3-chlorophenyl

H cyclopentyl I-23 4-chlorophenyl

H cyclohexyl I-24 3-fluorophenyl

H 4-methylcyclohexyl I-25 4-fluorophenyl

H ethyl I-26 3,4-dichlorophenyl

H tert-butyl I-27 3,4-difluorophenyl

H 2,2,2-trifluoroethyl I-28 4-trifluoromethylphenyl

H 1-methylcyclobutyl I-29 3-chlorophenyl

H 3-chloro-4-fluorophenyl I-30 4-chlorophenyl

H 3-chloro-5-fluorophenyl I-31 3-fluorophenyl

H 3-chlorophenyl I-32 4-fluorophenyl

H 4-chlorophenyl I-33 3,4-dichlorophenyl

H 3,5-dichlorophenyl I-34 3,4-difluorophenyl

H 3-chloro-4-fluorophenyl I-35 4-trifluoromethylphenyl

H 3-chloro-5-fluorophenyl I-36 3-chlorophenyl

H 3-trifluoromethylphenyl I-37 4-chlorophenyl

H 3-chlorophenyl I-38 3-chlorophenyl

—C(O)Me 3-chlorophenyl I-39 4-chlorophenyl

—C(O)Me 4-chlorophenyl I-40 3-fluorophenyl

—C(O)Me 3,5-dichlorophenyl I-41 4-fluorophenyl

—C(O)Me 3-chloro-4-fluorophenyl I-42 3-chlorophenyl

—CH₃ 3-chlorophenyl I-43 4-chlorophenyl

—CH₃ 4-chlorophenyl I-44 3-fluorophenyl

—CH₃ 3,5-dichlorophenyl I-45 4-fluorophenyl

—CH₃ 3-chloro-4-fluorophenyl I-46

H 3-chlorophenyl I-47

H 4-chlorophenyl I-48

H 3,5-dichlorophenyl I-49

H 3-chloro-4-fluorophenyl I-50

H 3-chloro-5-fluorophenyl I-51

H 3-trifluoromethylphenyl I-52

H 3-cyclopropylphenyl I-53

H 3-fluorophenyl I-54

H 4-fluorophenyl I-55

H 3-fluorobenzyl I-56 3-chlorophenyl

H 3-chlorophenyl I-57 4-chlorophenyl

H 4-chlorophenyl I-58 3-fluorophenyl

H 3,5-dichlorophenyl I-59 4-fluorophenyl

H 3-chloro-4-fluorophenyl I-60 3,4-dichlorophenyl

H 3-chloro-5-fluorophenyl I-61 3,4-difluorophenyl

H 3-trifluoromethylphenyl I-62 4-trifluoromethylphenyl

H 3-cyclopropylphenyl I-63 3-chlorophenyl

H 3-tert-butylphenyl I-64 4-chlorophenyl

H 2-cyclopropylphenyl I-65 3-chlorophenyl

H 3-chlorophenyl I-66 4-chlorophenyl

H 4-chlorophenyl I-67 3-fluorophenyl

H 3,5-dichlorophenyl I-68 4-fluorophenyl

H 3-chloro-4-fluorophenyl I-69 3,4-dichlorophenyl

H 3-chloro-5-fluorophenyl I-70 3,4-difluorophenyl

H 3-trifluoromethylphenyl

In certain other embodiments, the compound is one of the compoundslisted in Examples 1-4, or a pharmaceutically acceptable salt of saidcompounds. It is understood that the foregoing compounds can be combinedwith a pharmaceutically acceptable carrier to produce a pharmaceuticalcomposition.

C. Exemplary Procedures for Making Guanidine Compounds

Exemplary methods for preparing compounds described herein are providedin the examples. Further exemplary procedures for making variouscompounds described herein are described in Scheme 1 below. Thesynthetic scheme is provided for the purpose of illustrating theinvention, but not for limiting the scope or spirit of the invention.Starting materials can be obtained from commercial sources or beprepared based on procedures described in the literature.

The synthetic route in Scheme 1 involves reacting an optionallysubstituted benzoylchloride with potassium thiocyanate to form an acylisothiocyanate intermediate. This acyl isothiocyanate intermediate istreated with an amino-pyridone compound to form an acyl thiourea. Theacyl thiourea is reacted with1-ethyl-2′,2′-dimethylaminopropylcarbodiimide (EDCI) and a second aminecompound (e.g., an aniline or benzylamine) to form the desired pyridonylguanidine compound. To the extent either the amino-pyridone or thesecond amine compound contain a further functional group that mayundergo reaction under the conditions illustrated in Scheme 1, standardprotecting group strategies for protection and deprotection may beemployed. See, for example, Greene, T. W.; Wuts, P. G. M. ProtectiveGroups in Organic Synthesis, 2^(nd) ed.; Wiley: New York, 1991. Itunderstood that the optionally substituted benzoylchloride startingmaterial can be replaced with a heteroaryl acid chloride (i.e.,nicotinoyl chloride) to prepare pyridonyl guanidines containing a—C(O)-heteroaryl moiety.

III. Therapeutic Applications of Pyridonyl Guanidine Compounds

It is contemplated that the guanidine compounds described herein, suchas the guanidine compounds of Formula I, I-A, and I-B, providetherapeutic benefits to patients suffering from any one or more of anumber of conditions, e.g., diseases characterized by dysregulation ofF₁F₀-ATPase activity, diseases characterized by dysregulation ofnecrosis and/or apoptosis processes in a cell or tissue, diseasecharacterized by aberrant cell growth and/or hyperproliferation. Thecompounds described herein can also be used to treat a variety ofdysregulatory disorders related to cellular death as described elsewhereherein. Additionally, the compounds described herein can be used toinhibit ATP synthesis.

Accordingly, one aspect of the invention provides a method of treating asubject suffering from a medical disorder. The method comprisesadministering to the subject a therapeutically effective amount of oneor more pyridonyl guanidine compounds described herein, e.g., a compoundof Formula I as described in Section II, in order to alleviate a symptomof the disorder.

A large number of medical disorders can be treated using the guanidinecompounds described herein. For example, the compounds described hereincan be used to treat medical disorders characterized by dysregulation ofnecrosis and/or apoptosis processes in a cell or tissue, diseasescharacterized by aberrant cell growth and/or hyperproliferation, etc.,or lupus, rheumatoid arthritis, psoriasis, graft-versus-host disease,Crohn's disease, inflammatory bowel disease, multiple sclerosis,cardiovascular disease, myeloma, lymphoma, cancer, and bacterialinfection. In certain embodiments, the cancer is a solid tumor,leukemia, colon cancer, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, lungcancer, small cell lung cancer, non-small cell lung cancer, bladdercancer, stomach cancer, cervical cancer, testicular tumor, skin cancer,rectal cancer, thyroid cancer, kidney cancer, uterus cancer, espophaguscancer, liver cancer, an acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, or retinoblastoma.

Although not wishing to be bound to a particular theory, it is believedthat the compounds impart therapeutic benefit by modulating (e.g.,inhibiting or promoting) the activity of the F₁F₀-ATPase complexes(e.g., mitochondrial F₁F₀-ATPase complexes) in affected cells ortissues. In some embodiments, the compositions of the present inventionare used to treat immune/chronic inflammatory conditions (e.g.,psoriasis, autoimmune disorders, organ-transplant rejection, andepidermal hyperplasia). In further embodiments, the compositions of thepresent invention are used in conjunction with stenosis therapy to treatcompromised (e.g., occluded) vessels.

In certain embodiments, a composition comprising a guanidine compound isadministered under conditions (e.g., timing, dose, co-administrationwith other agent, mode of administration, selection of subject, use oftargeting agents, etc.) that maximize desired effects directed at theF₁F₀-ATPase.

In certain embodiments, the medical disorder is an immune disorder. Incertain other embodiments, the medical disorder is an inflammatorydisorder. In certain other embodiments, the medical disorder is anautoimmune disorder. In certain other embodiments, the medical disorderis rheumatoid arthritis, psoriasis, chronic graft-versus-host disease,acute graft-versus-host disease, Crohn's disease, inflammatory boweldisease, multiple sclerosis, systemic lupus erythematosus, Celiac Sprue,idiopathic thrombocytopenic thrombotic purpura, myasthenia gravis,Sjogren's syndrome, scleroderma, ulcerative colitis, asthma, uveitis, orepidermal hyperplasia.

In certain other embodiments, the medical disorder is cartilageinflammation, bone degradation, arthritis, juvenile arthritis, juvenilerheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis,polyarticular juvenile rheumatoid arthritis, systemic onset juvenilerheumatoid arthritis, juvenile ankylosing spondylitis, juvenileenteropathic arthritis, juvenile reactive arthritis, juvenile Reter'sSyndrome, SEA Syndrome, juvenile dermatomyositis, juvenile psoriaticarthritis, juvenile scleroderma, juvenile systemic lupus erythematosus,juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticularrheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosingspondylitis, enteropathic arthritis, reactive arthritis, Reter'sSyndrome, dermatomyositis, psoriatic arthritis, vasculitis, myolitis,polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis nodossa,Wegener's granulomatosis, arteritis, polymyalgia rheumatica,sarcoidosis, sclerosis, primary biliary sclerosis, sclerosingcholangitis, dermatitis, atopic dermatitis, atherosclerosis, Still'sdisease, chronic obstructive pulmonary disease, Guillain-Barre disease,Type I diabetes mellitus, Graves' disease, Addison's disease, Raynaud'sphenomenon, or autoimmune hepatitis. In certain embodiments, thepsoriasis is plaque psoriasis, guttate psoriasis, inverse psoriasis,pustular psoriasis, or erythrodermic psoriasis.

In certain other embodiments, the medical disorder is Crohn's disease,inflammatory bowel disease, multiple sclerosis, graft-versus-hostdisease, lupus, rheumatoid arthritis, or psoriasis. In certain otherembodiments, the medical disorder is cardiovascular disease, myeloma,lymphoma, or cancer. In certain other embodiments, the medical disorderis lupus, rheumatoid arthritis, psoriasis, graft-versus-host disease,myeloma, or lymphoma. In certain other embodiments, the medical disorderis cardiovascular disease or cancer. In certain other embodiments, themedical disorder is Crohn's disease, inflammatory bowel disease, ormultiple sclerosis. In certain other embodiments, the medical disorderis graft-versus-host disease. In further embodiments, the medicaldisorder is a bacterial infection. In certain embodiments, the patient(or subject) is a human.

As indicated above, the guanidine compounds described herein can be usedin the treatment of a bacterial infection. A variety of bacteria arecontemplated to be susceptible to the guanidine compounds.Representative bacteria include Staphylococci species, e.g., S. aureus;Enterococci species, e.g., E. faecalis and E. faecium; Streptococcispecies, e.g., S. pyogenes and S. pneumoniae; Escherichia species, e.g.,E. coli, including enterotoxigenic, enteropathogenic, enteroinvasive,enterohemorrhagic and enteroaggregative E. coli strains; Haemophilusspecies, e.g., H. influenza; and Moraxella species, e.g., M.catarrhalis. Other examples include Mycobacteria species, e.g., M.tuberculosis, M. avian-intracellulare, M. kansasii, M. Bovis, M.africanum, M. genavense, M. leprae, M. xenopi, M. simiae, M.scrofulaceum, M. malmoense, M. celatum, M. abscessus, M. chelonae, M.szulgai, M. gordonae, M. haemophilum, M. fortuni and M. marinum;Corynebacteria species, e.g., C. diphtheriae; Vibrio species, e.g., V.cholerae; Campylobacter species, e.g., C. jejuni; Helicobacter species,e.g., H. pylori; Pseudomonas species, e.g., P. aeruginosa; Legionellaspecies, e.g., L. pneumophila; Treponema species, e.g., T. pallidum;Borrelia species, e.g., B. burgdorferi; Listeria species, e.g., L.monocytogenes; Bacillus species, e.g., B. cereus; Bordatella species,e.g., B. pertussis; Clostridium species, e.g., C. perfringens, C.tetani, C. difficile and C. botulinum; Neisseria species, e.g., N.meningitidis and N. gonorrhoeae; Chlamydia species, e.g., C. psittaci,C. pneumoniae and C. trachomatis; Rickettsia species, e.g., R.rickettsii and R. prowazekii; Shigella species, e.g., S. sonnei;Salmonella species, e.g., S. typhimurium; Yersinia species, e.g., Y.enterocolitica and Y. pseudotuberculosis; Klebsiella species, e.g., K.pneumoniae; Mycoplasma species, e.g., M. pneumoniae; and Trypanosomabrucei. In certain embodiments, the guanidine compounds described hereinare used to treat a subject suffering from a bacterial infectionselected from the group consisting of S. aureus, E. faecalis, E.faecium, S. pyogenes, S. pneumonia, and P. aeruginosa. In certainembodiments, the guanidine compounds described herein are used to treata subject suffering from a Trypanosoma brucei infection.

The antibacterial activity of the compounds described herein may beevaluated using standard assays known in the art, such as the microbrothdilution minimum inhibition concentration (MIC) assay, as furtherdescribed in National Committee for Clinical Laboratory Standards.Performance Standards for Antimicrobial Susceptibility Testing;Fourteenth Informational Supplement. NCCLS document M100-S14 {ISBN1-56238-516-X}. This assay may be used to determine the minimumconcentration of a compound necessary to prevent visible bacterialgrowth in a solution. In general, the drug to be tested is seriallydiluted into wells, and aliquots of liquid bacterial culture are added.This mixture is incubated under appropriate conditions, and then testedfor growth of the bacteria. Compounds with low or no antibiotic activity(a high MIC) will allow growth at high concentrations of compound, whilecompounds with high antibiotic activity will allow bacterial growth onlyat lower concentrations (a low MIC).

The assay uses stock bacterial culture conditions appropriate for thechosen strain of bacteria. Stock cultures from the permanent stockculture collection can be stored as frozen suspensions at −70° C.Cultures may be suspended in 10% skim milk (BD) prior to snap freezingin dry ice/ethanol and then placed in a −70° C. freezer. Cultures may bemaintained on Tryptic Soy Agar containing 5% Sheep Blood at roomtemperature (20° C.), and each culture may be recovered from frozen formand transferred an additional time before MIC testing. Fresh plates areinoculated the day before testing, incubated overnight, and checked toconfirm purity and identity.

The identity and purity of the cultures recovered from the stock culturecan be confirmed to rule out the possibility of contamination. Theidentity of the strains may be confirmed by standard microbiologicalmethods (See, e.g., Murray et al., Manual of Clinical Microbiology,Eighth Edition. ASM Press {ISBN 1-55581-255-4}). In general, culturesare streaked onto appropriate agar plates for visualization of purity,expected colony morphology, and hemolytic patterns. Gram stains can alsobe utilized. The identities are confirmed using a MicroScan WalkAway 40SI Instrument (Dade Behring, West Sacramento, Calif.). This deviceutilizes an automated incubator, reader, and computer to assess foridentification purposes the biochemical reactions carried out by eachorganism. The MicroScan WalkAway can also be used to determine apreliminary MIC, which may be confirmed using the method describedbelow.

Frozen stock cultures may be used as the initial source of organisms forperforming microbroth dilution minimum inhibition concentration (MIC)testing. Stock cultures are passed on their standard growth medium forat least 1 growth cycle (18-24 hours) prior to their use. Most bacteriamay be prepared directly from agar plates in 10 mL aliquots of theappropriate broth medium. Bacterial cultures are adjusted to the opacityof a 0.5 McFarland Standard (optical density value of 0.28-0.33 on aPerkin-Elmer Lambda EZ150 Spectrophotometer, Wellesley, Mass., set at awavelength of 600 nm). The adjusted cultures are then diluted 400 fold(0.25 mL inoculum+100 mL broth) in growth media to produce a startingsuspension of approximately 5×105 colony forming units (CFU)/mL. Mostbacterial strains may be tested in cation adjusted Mueller Hinton Broth(CAMHB).

Test compounds (“drugs”) are solubilized in a solvent suitable for theassay, such as DMSO. Drug stock solutions may be prepared on the day oftesting. Microbroth dilution stock plates may be prepared in twodilution series, 64 to 0.06 μg drug/mL and 0.25 to 0.00025 μg drug/mL.For the high concentration series, 200 μL of stock solution (2 mg/mL) isadded to duplicate rows of a 96-well microtiter plate. This is used asthe first well in the dilution series. Serial two-fold decrementaldilutions are made using a BioMek FX robot (Beckman Coulter Inc.,Fullerton, Calif.) with 10 of the remaining 11 wells, each of which willcontain 100 μL of the appropriate solvent/diluent. Row 12 containssolvent/diluent only and serves as the control. For the first well ofthe low concentration series, 200 μL of an 8 μg/mL stock are added toduplicate rows of a 96-well plate. Serial two-fold dilutions are made asdescribed above.

Daughter 96-well plates may be spotted (3.2 μL/well) from the stockplates listed above using the BioMek FX robot and used immediately orfrozen at −70° C. until use. Aerobic organisms are inoculated (100 μLvolumes) into the thawed plates using the BioMek FX robot. Theinoculated plates are be placed in stacks and covered with an emptyplate. These plates are then incubated for 16 to 24 hours in ambientatmosphere according to CLSI guidelines (National Committee for ClinicalLaboratory Standards, Methods for Dilution, Antimicrobial Tests forBacteria that Grow Aerobically; Approved Standard-Sixth Edition. NCCLSdocument M7-A6 {ISBN 1-56238-486-4}).

After inoculation and incubation, the degree of bacterial growth can beestimated visually with the aid of a Test Reading Mirror (DynexTechnologies 220 16) in a darkened room with a single light shiningdirectly through the top of the microbroth tray. The MIC is the lowestconcentration of drug that prevents macroscopically visible growth underthe conditions of the test.

Additionally, any one or more of the pyridonyl guanidine compoundsdescribed herein can be used to treat a F₁F₀-ATP hydrolase associateddisorder (e.g., myocardial infarction, ventricular hypertrophy, coronaryartery disease, non-Q wave MI, congestive heart failure, cardiacarrhythmias, unstable angina, chronic stable angina, Prinzmetal'sangina, high blood pressure, intermittent claudication, peripheralocclusive arterial disease, thrombotic or thromboembolic symptoms ofthromboembolic stroke, venous thrombosis, arterial thrombosis, cerebralthrombosis, pulmonary embolism, cerebral embolism, thrombophilia,disseminated intravascular coagulation, restenosis, atrial fibrillation,ventricular enlargement, atherosclerotic vascular disease,atherosclerotic plaque rupture, atherosclerotic plaque formation,transplant atherosclerosis, vascular remodeling atherosclerosis, cancer,surgery, inflammation, systematic infection, artificial surfaces,interventional cardiology, immobility, medication, pregnancy and fetalloss, and diabetic complications comprising retinopathy, nephropathy andneuropathy) in a subject.

Combination Therapy

Additionally, the guanidine compounds described herein can be used incombination with at least one other therapeutic agent, such as Bz-423 (abenzodiazepine compound as described in U.S. Pat. Nos. 7,144,880 and7,125,866, U.S. patent application Ser. Nos. 11/586,097, 11/585,492,11/445,010, 11/324,419, 11/176,719, 11/110,228, 10/935,333, 10/886,450,10/795,535, 10/634,114, 10/427, 211, 10/217,878, and 09/767,283, andU.S. Provisional Patent Nos. 60/878,519, 60/812,270, 60/802,394,60/732,045, 60/730,711, 60/704,102, 60/686,348, 60/641,040, 60/607,599,and 60/565,788), potassium channel openers, calcium channel blockers,sodium hydrogen exchanger inhibitors, antiarrhythmic agents,antiatherosclerotic agents, anticoagulants, antithrombotic agents,prothrombolytic agents, fibrinogen antagonists, diuretics,antihypertensive agents, ATPase inhibitors, mineralocorticoid receptorantagonists, phosphodiesterase inhibitors, antidiabetic agents,anti-inflammatory agents, antioxidants, angiogenesis modulators,antiosteoporosis agents, hormone replacement therapies, hormone receptormodulators, oral contraceptives, antiobesity agents, antidepressants,antianxiety agents, antipsychotic agents, antiproliferative agents,antitumor agents, antiulcer and gastroesophageal reflux disease agents,growth hormone agents and/or growth hormone secretagogues, thyroidmimetics, anti-infective agents, antiviral agents, antibacterial agents,antifungal agents, cholesterol/lipid lowering agents and lipid profiletherapies, and agents that mimic ischemic preconditioning and/ormyocardial stunning, antiatherosclerotic agents, anticoagulants,antithrombotic agents, antihypertensive agents, antidiabetic agents, andantihypertensive agents selected from ACE inhibitors, AT-1 receptorantagonists, ET receptor antagonists, dual ET/AII receptor antagonists,vasopepsidase inhibitors, an antiplatelet agent selected from GPIIb/IIIablockers, P2Y₁ and P2Y₁₂ antagonists, thromboxane receptor antagonists,or aspirin, along with a pharmaceutically-acceptable carrier or diluentin a pharmaceutical composition.

IV. Pharmaceutical Compositions, Formulations, and ExemplaryAdministration Routes and Dosing Considerations

Exemplary embodiments of various contemplated medicaments andpharmaceutical compositions are provided below.

A. Preparing Medicaments

Compounds of the present invention are useful in the preparation ofmedicaments to treat a variety of conditions, such as conditionsassociated with dysregulation of cell death, aberrant cell growth andhyperproliferation. One of skill in the art will appreciate that any oneor more of the compounds described herein, including the many specificembodiments, are prepared by applying standard pharmaceuticalmanufacturing procedures. Such medicaments can be delivered to thesubject by using delivery methods that are well-known in thepharmaceutical arts.

B. Exemplary Pharmaceutical Compositions and Formulation

In some embodiments of the present invention, the compositions areadministered alone, while in some other embodiments, the compositionsare preferably present in a pharmaceutical formulation comprising atleast one active ingredient/agent, as discussed above, together with asolid support or alternatively, together with one or morepharmaceutically acceptable carriers and optionally other therapeuticagents (e.g., those described in section III hereinabove). Each carriershould be “acceptable” in the sense that it is compatible with the otheringredients of the formulation and not injurious to the subject.

Contemplated formulations include those suitable for oral, rectal,nasal, topical (including transdermal, buccal and sublingual), vaginal,parenteral (including subcutaneous, intramuscular, intravenous andintradermal) and pulmonary administration. In some embodiments,formulations are conveniently presented in unit dosage form and areprepared by any method known in the art of pharmacy. Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association (e.g., mixing) the active ingredient withliquid carriers or finely divided solid carriers or both, and then ifnecessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,wherein each preferably contains a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. In other embodiments, the activeingredient is presented as a bolus, electuary, or paste, etc.

In some embodiments, tablets comprise at least one active ingredient andoptionally one or more accessory agents/carriers are made by compressingor molding the respective agents. In some embodiments, compressedtablets are prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g., povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose) surface-active ordispersing agent. Molded tablets are made by molding in a suitablemachine a mixture of the powdered compound (e.g., active ingredient)moistened with an inert liquid diluent. Tablets may optionally be coatedor scored and may be formulated so as to provide slow or controlledrelease of the active ingredient therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile. Tablets may optionally be provided with anenteric coating, to provide release in parts of the gut other than thestomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention are optionally formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosolsor oils. In alternative embodiments, topical formulations comprisepatches or dressings such as a bandage or adhesive plasters impregnatedwith active ingredient(s), and optionally one or more excipients ordiluents. In some embodiments, the topical formulations include acompound(s) that enhances absorption or penetration of the activeagent(s) through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethylsulfoxide (DMSO) andrelated analogues.

If desired, the aqueous phase of a cream base includes, for example, atleast about 30% w/w of a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof.

In some embodiments, oily phase emulsions of this invention areconstituted from known ingredients in a known manner. This phasetypically comprises a lone emulsifier (otherwise known as an emulgent),it is also desirable in some embodiments for this phase to furthercomprise a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil.

Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier so as to act as a stabilizer. In some embodimentsit is also preferable to include both an oil and a fat. Together, theemulsifier(s) with or without stabilizer(s) make up the so-calledemulsifying wax, and the wax together with the oil and/or fat make upthe so-called emulsifying ointment base which forms the oily dispersedphase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired properties (e.g., cosmetic properties), since thesolubility of the active compound/agent in most oils likely to be usedin pharmaceutical emulsion formulations is very low. Thus creams shouldpreferably be non-greasy, non-staining and washable products withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used, the last three being preferredesters. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils can beused.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration may be presented as a suppositorywith suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, creams, gels, pastes, foams or spray formulations containingin addition to the agent, such carriers as are known in the art to beappropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include coarse powders having a particle size, for example, inthe range of about 20 to about 500 microns which are administered in themanner in which snuff is taken, i.e., by rapid inhalation (e.g., forced)through the nasal passage from a container of the powder held close upto the nose. Other suitable formulations wherein the carrier is a liquidfor administration include, but are not limited to, nasal sprays, drops,or aerosols by nebulizer, and include aqueous or oily solutions of theagents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. In some embodiments, the formulations arepresented/formulated in unit-dose or multi-dose sealed containers, forexample, ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets of the kind previouslydescribed.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents. It also is intended that the agents, compositions and methods ofthis invention be combined with other suitable compositions andtherapies. Still other formulations optionally include food additives(suitable sweeteners, flavorings, colorings, etc.), phytonutrients(e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, andother acceptable compositions (e.g., conjugated linoelic acid),extenders, and stabilizers, etc.

C. Exemplary Administration Routes and Dosing Considerations

Various delivery systems are known and can be used to administertherapeutic agents (e.g., exemplary compounds as described above) of thepresent invention, e.g., encapsulation in liposomes, microparticles,microcapsules, receptor-mediated endocytosis, and the like. Methods ofdelivery include, but are not limited to, intra-arterial,intra-muscular, intravenous, intranasal, and oral routes. In specificembodiments, it may be desirable to administer the pharmaceuticalcompositions of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, injection, or by means of a catheter.

The agents identified can be administered to subjects or individualssusceptible to or at risk of developing pathological growth of targetcells and correlated conditions. When the agent is administered to asubject such as a mouse, a rat or a human patient, the agent can beadded to a pharmaceutically acceptable carrier and systemically ortopically administered to the subject. To identify patients that can bebeneficially treated, a tissue sample is removed from the patient andthe cells are assayed for sensitivity to the agent.

Therapeutic amounts are empirically determined and vary with thepathology being treated, the subject being treated and the efficacy andtoxicity of the agent. When delivered to an animal, the method is usefulto further confirm efficacy of the agent. One example of an animal modelis MLR/MpJ-lpr/lpr (“MLR-lpr”) (available from Jackson Laboratories, BarHarbor, Me.). MLR-lpr mice develop systemic autoimmune disease.Alternatively, other animal models can be developed by inducing tumorgrowth, for example, by subcutaneously inoculating nude mice with about10⁵ to about 10⁹ hyperproliferative, cancer or target cells as definedherein. When the tumor is established, the compounds described hereinare administered, for example, by subcutaneous injection around thetumor. Tumor measurements to determine reduction of tumor size are madein two dimensions using venier calipers twice a week. Other animalmodels may also be employed as appropriate. Such animal models for theabove-described diseases and conditions are well-known in the art.

In some embodiments, in vivo administration is effected in one dose,continuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are well known to those of skill in the art and vary withthe composition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations are carried out with the dose level and pattern beingselected by the treating physician.

Suitable dosage formulations and methods of administering the agents arereadily determined by those of skill in the art. Preferably, thecompounds are administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg. When the compounds describedherein are co-administered with another agent (e.g., as sensitizingagents), the effective amount may be less than when the agent is usedalone.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor non-aqueous diluents, syrups, granulates or powders. In addition toan agent of the present invention, the pharmaceutical compositions canalso contain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

More particularly, an agent of the present invention also referred toherein as the active ingredient, may be administered for therapy by anysuitable route including, but not limited to, oral, rectal, nasal,topical (including, but not limited to, transdermal, aerosol, buccal andsublingual), vaginal, parental (including, but not limited to,subcutaneous, intramuscular, intravenous and intradermal) and pulmonary.It is also appreciated that the preferred route varies with thecondition and age of the recipient, and the disease being treated.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of disease. This may be achieved, forexample, by the intravenous injection of the agent, optionally insaline, or by oral administration, for example, as a tablet, capsule orsyrup containing the active ingredient.

Desirable blood levels of the agent may be maintained by a continuousinfusion to provide a therapeutic amount of the active ingredient withindisease tissue. The use of operative combinations is contemplated toprovide therapeutic combinations requiring a lower total dosage of eachcomponent than may be required when each individual therapeutic compoundor drug is used alone, thereby reducing adverse effects.

D. Exemplary Co-administration Routes and Dosing Considerations

The invention also includes methods involving co-administration of thecompounds described herein with one or more additional active agents.Indeed, it is a further aspect of this invention to provide methods forenhancing prior art therapies and/or pharmaceutical compositions byco-administering a compound of this invention. In co-administrationprocedures, the agents may be administered concurrently or sequentially.In one embodiment, the compounds described herein are administered priorto the other active agent(s). The pharmaceutical formulations and modesof administration may be any of those described above. In addition, thetwo or more co-administered chemical agents, biological agents orradiation may each be administered using different modes or differentformulations.

The agent or agents to be co-administered depend on the type ofcondition being treated. For example, when the condition being treatedis cancer, the additional agent can be a chemotherapeutic agent orradiation. When the condition being treated is an immune disorder, theadditional agent can be an immunosuppressant or an anti-inflammatoryagent. When the condition being treated is chronic inflammation, theadditional agent can be an anti-inflammatory agent. The additionalagents to be co-administered, such as anticancer, immunosuppressant,anti-inflammatory, can be any of the well-known agents in the art,including, but not limited to, those that are currently in clinical use.The determination of appropriate type and dosage of radiation treatmentis also within the skill in the art or can be determined with relativeease.

Treatment of the various conditions associated with abnormal apoptosisis generally limited by the following two major factors: (1) thedevelopment of drug resistance and (2) the toxicity of known therapeuticagents. In certain cancers, for example, resistance to chemicals andradiation therapy has been shown to be associated with inhibition ofapoptosis. Some therapeutic agents have deleterious side effects,including non-specific lymphotoxicity, renal and bone marrow toxicity.

The methods described herein address both these problems. Drugresistance, where increasing dosages are required to achieve therapeuticbenefit, is overcome by co-administering the compounds described hereinwith the known agent. The compounds described herein sensitize targetcells to known agents (and vice versa) and, accordingly, less of theseagents are needed to achieve a therapeutic benefit.

The sensitizing function of the claimed compounds also addresses theproblems associated with toxic effects of known therapeutics. Ininstances where the known agent is toxic, it is desirable to limit thedosages administered in all cases, and particularly in those cases wheredrug resistance has increased the requisite dosage. When the claimedcompounds are co-administered with the known agent, they reduce thedosage required which, in turn, reduces the deleterious effects.

EXAMPLES

The invention now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 General Methods for Preparing Pyridonyl Guanidine Compounds

Described below are exemplary, general synthetic procedures for makingpyridonyl guanidine compounds, along with an exemplary syntheticprocedure for making the specific pyridonyl guanidine compound(Z)-3,4-difluoro-N-((5-hydroxypyridin-3-ylamino)(3-(trifluoromethyl)phenylamino)methylene)benzamide.

Part I: General Method for Making Pyridonyl Guandine Compounds

Guanidines can be prepared from an acid chloride, a first amine, and asecond amine using a three-step procedure. First, the requisite acidchloride is combined with potassium thiocyanate in an organic solvent,and this mixture is stirred at ambient temperature for 1-4 hours. Theresulting mixture is concentrated in vacuo and used immediately.

In a second step, an appropriate first amine (RNH₂) is dissolved in anorganic solvent, such as methylene chloride, at ambient temperature andthe acyl isothiocyanate from the first step is added. The resultingmixture is stirred at ambient temperature for 8-16 hours. The solventsare evaporated in vacuo and the resulting residue treated with a warmnon-polar organic solvent, then allowed to cool and collected byfiltration. The collected residue is rinsed with a non-polar organicsolvent and dried. The resulting residue can be used without furtherpurification. Alternatively, the first amine in the form of ahydrochloride salt is dissolved in an organic solvent and treated with ahindered organic base such as triethylamine then stirred at ambienttemperature for 1-4 hours. The acyl isothiocyanate from step 1 is thenadded and the reaction mixture stirred at ambient temperature for 8-16hours. The solvents are removed in vacuo and the resulting residue ispurified by chromatography.

In the third step, the acyl thiourea from step 2 and an appropriatesecond amine (R¹—NH₂) are dissolved in a polar organic solvent such asdimethylformamide at ambient temperature to form a mixture. To thismixture, 1-ethyl-2′,2′-dimethylaminopropylcarbodiimide is added and theresulting mixture is stirred until the reaction appears complete by HPLCanalysis of aliquots of the reaction mixture. Typical reaction timesrange from 30 minutes to 12 hours, and the reaction mixture may beheated (e.g., to approximately 60° C.) to accelerate the reaction. Oncethe reaction appears to be complete by HPLC analysis, the reactionmixture is diluted with an organic solvent (such as ethylacetate),washed with water, washed with brine, and the organic layer is driedover an appropriate drying agent, filtered, and the solvents removedunder reduced pressure. The desired product can be purified bychromatography if necessary.

Part II: Exemplary Synthetic Procedure for Preparing Pyridonyl GuanidineCompound(Z)-3,4-Difluoro-N-((5-hydroxypyridin-3-ylamino)(3-(trifluoromethyl)phenylamino)methylene)benzamide Step A: Representative Procedure for Preparing aSubstituted Benzoyl Isothiocyanate in Situ and Conversion to a Thiourea

To a solution of 3,4-difluorobenzoyl chloride (5.00 mL, 39 7 mmol, 1equiv) in acetonitrile (79 mL) at 0° C. was added solid potassiumthioisocyanate (4.25 g, 43.7 mmol, 1.10 equiv.). After 10 minutes thecooling bath was removed and the reaction mixture was allowed to warm upto room temperature. The reaction mixture was stirred at roomtemperature for 1 h. Neat 3-trifluoromethylaniline (6.40 g, 39.7 mmol,1.00 equiv.) was added and the reaction mixture was stirred at roomtemperature for 18 h. The solution was diluted with water (250 mL) andthe resulting suspension was filtered and dried in vacuo at 70° C. toafford 14.06 g ofN1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine.

Step B: Representative Procedure for the Coupling of a Thiourea to anAmine

N1-((Ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine (43 mg,0.278 mmol),3,4-difluoro-N-((3-(trifluoromethyl)phenyl)carbamothioyl)benzamide (100mg, 0.278 mmol) and 5-aminopyridin-3-ol (31 mg, 0.278 mmol) and werecombined in a glass vial and dry dimethylformamide (1.5 mL) was added.The vial was capped and the mixture was heated at 65° C. for 2 h. Aftercooling the mixture to room temperature, the mixture was diluted withethyl acetate and washed with water then brine. The organic layer wasevaporated onto silica gel and the product was purified bychromatography eluting with 50-100% ethyl acetate in hexanes to give(Z)-3,4-difluoro-N-(((5-hydroxypyridin-3-yl)amino)((3(trifluoromethyl)phenyl)amino)methylene)benzamide as a colorless oil.

Example 2 Preparation of(E)-N—((N-(3-Chloro-4-Fluorobenzyl)Acetamido)(6-Hydroxypyridin-2-Ylamino)Methylene)-4-(Trifluoromethyl)Benzamide

Pyridonyl guanidine compound(Z)—N-(((3-chloro-4-fluorobenzyl)amino)((6-hydroxypyridin-2-yl)amino)methylene)-4-(trifluoromethyl)benzamidewas prepared based on the procedures described in Example 1. Then,(Z)-N-(((3-chloro-4-fluorobenzyl)amino)((6-hydroxypyridin-2-yl)amino)methylene)-4-(trifluoromethyl)benzamide(0.1 g, 0.214 mmol) was dissolved in pyridine (1 mL) with4-dimethylaminopyridine (0.026 g, 0.214 mmol) under nitrogen at roomtemperature and acetic anhydride was added (0.022 g, 0.214 mmol). Thereaction mixture was stirred at room temperature for 4 h forming a thickwhite slurry. Analysis by RP-HPLC indicated a ˜1:1 mixture of startingmaterial and product. Additional pyridine (approximately 0.5 mL) andacetic anhydride (˜10 uL) were added. Stirring was continued for another4 h then the mixture was diluted with water and ethyl acetate. Saturatedaqueous NH₄Cl was added to the mixture and the layers were separated.The organic layer was washed with water then brine and dried (MgSO₄).Chromatography, eluting with 30-50% ethyl acetate in hexanes, gave awhite solid which was triturated with diethyl ether to provide(E)-N—((N-(3-chloro-4-fluorobenzyl)acetamido)((6-hydroxypyridin-2-yl)amino)methylene)-4-(trifluoromethyl)benzamide(52 mg, 48%).

Example 3 Preparation of Sodium(Z)-6-(3-(2-Cyclopropyl-4-Fluorophenyl)-2-(4-(Trifluoromethyl)Benzoyl)Guanidino)Pyridin-2-YlPhosphate

The title compound was prepared from pyridonyl guanidine compound(Z)-N-(((3-chloro-4-fluorobenzyl)amino)((6-hydroxypyridin-2-yl)amino)methylene)-4-(trifluoromethyl)benzamide,which was prepared based on the procedures described in Example 1.

Step 1: Installation of a Phosphoric Acid Group

(Z)-N-(((2-Cyclopropyl-4-fluorophenyl)amino)((6-hydroxypyridin-2-yl)amino)methylene)-4-(trifluoromethyl)benzamide(50 mg, 0.11 mmol) was dissolved in THF (4.5 mL) and pyridine (70 μL,0.87 mmol) was added. The reaction was then cooled to 0° C. andphosphorousoxy chloride (41 μL, 0.44 mmol) was added dropwise. Thereaction was stirred for 1 h at 0° C., then it was quenched with 1N NaOH(to pH=12). The aqueous phase was diluted (3 mL water), then acidifiedto pH=1 with 6 N HCl. The aqueous phase was then extracted five timesinto 1:1 EtOAc:THF. The combined extracts were dried over sodiumsulfate, then concentrated to provide the crude product. The crudeproduct was purified by preparative HPLC (water/MeOH) providing(Z)-6-(3-(2-cyclopropyl-4-fluorophenyl)-2-(4-(trifluoromethyl)benzoyl)guanidino)pyridin-2-yldihydrogen phosphate (13 mg, 0.024 mmol, 22% yield). ¹HNMR (400 MHz,DMSO-d⁶) δ 13.08 (bs, 1H), 11.79 (bs, 1H), 11.09 (bs, 1H), 10.64 (bs,1H), 8.07-8.02 (m, 1H), 7.94-7.87 (m, 1H), 7.74 (d, 1H, J=7.6 Hz), 7.57(m, 1H), 7.31 (dd, 1H, J=8.0, 6.6), 7.10-7.02 (m, 2H), 6.91-6.82 (m,3H), 6.73 (d, 1H, J=8.0 Hz), 2.09-1.93 (m, 1H), 0.92-0.88 (m, 2H),0.74-0.70 (m, 1H), 0.67-0.63 (m, 1H). MS (ES+) 538.8.

Step 2: Conversion of the Phosphoric Acid Group to a Sodium PhosphateGroup

(Z)-6-(3-(2-Cyclopropyl-4-fluorophenyl)-2-(4-(trifluoromethyl)benzoyl)guanidine)pyridine-2-yl dihydrogen phosphate (13 mg, 0.024 mmol) wasdissolved in THF (5 mL), and 48 mL of 2 N sodium hydroxide solution(0.048 mmol) was added. The solution was concentrated in vacuo toprovide the title compound as a yellow solid.

Example 4 Preparation of(Z)—N-(((6-(Methylsulfonamido)Pyridine-2-Yl)Amino)(2-(Trifluoromethyl)Benzyl)Amino)Methylene)-4-(Trifluoromethyl)Benzamide

The title compound was prepared according to the procedures describedbelow in Steps 1 and 2.

Step 1: Preparation of(Z)—N-(((6-Aminopyridin-2-yl)amino)((2-(trifluoromethyl)benzyl)amino)methylene)-4-(trifluoromethyl)benzamide

A solution of4-(trifluoromethyl)-N-((2-(trifluoromethyl)benzyl)carbamothioyl)benzamide (2.0 g, 4.92 mmol), 2,6-diaminopyridine (591 mg, 5.41 mmol),and 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDChydrochloride) (1.13 g, 5.91 mmol) in DMF (49 mL) was heated to 65° C.for 30 minutes, then allowed to cool to room temperature. The mixturewas then diluted with ethyl acetate, washed with aqueous sodiumbicarbonate solution, washed with brine, dried over sodium sulfate, andconcentrated to provide the crude product. The crude product waspurified by chromatography (gradient: 9:1 hexanes:EtOAc to 6:4hexanes:EtOAc) to provide(Z)—N-(((6-aminopyridin-2-yl)amino)((2-(trifluoromethyl)benzyl)amino)methylene)-4-(trifluoromethyl)benzamide as a yellow solid(1.31 g, 55% yield).

Step 2: Preparation of(Z)—N-(((6-(Methylsulfonamido)pyridine-2-yl)amino)((2-(trifluoromethyl)benzyl)amino)methylene)-4-(trifluoromethyl)benzamide

(Z)-N-(((6-Aminopyridin-2-yl)amino)((2-(trifluoromethyl)benzyl)amino)methylene)-4-(trifluoromethyl)benzamide (200 mg, 0.42 mmol) wasdissolved in dichloromethane (3.8 mL) and pyridine (0.4 mL) was addedfollowed by mesyl chloride (32 μL, 0.42 mmol). The reaction was stirredat room temperature for 3 days, and another equivalent of mesyl chloride(32 μL, 0.42 mmol) was added, and stirring continued for another 24 h.The crude material then was concentrated, and the residue waspartitioned between water and ethyl acetate, and the organic layer waswashed with brine, then dried over sodium sulfate, and concentrated invacuo to provide the crude product. The crude product was purified bychromatography (gradient: 8:2 hexanes:EtOAc to 2:8 hexanes:EtOAc) toprovide the title compound (95 mg, 41% yield).

Example 5 Pyridonyl Guanidine Compounds & Characterization Data

Compounds in Table 2 below were prepared based on the proceduresdescribed in Examples 1-4 and procedures described in the detaileddescription. Starting materials can be obtained from commercial sources(e.g., acid chloride: 4-chlorobenzoyl chloride, 3-chlorobenzoylchloride, and 4-fluorobenzoyl chloride; first amine compound:2-trifluoromethylaniline, 2-tert-butylaniline; and second amine:6-aminopyridin-3-ol, 5-aminopyridin-5-ol, and 6-aminopyridin-2-ol) orreadily prepared from commercially available materials. Furthermore,exemplary compounds were characterized by high performance liquidchromatography (HPLC), mass spectrometry (MS) and/or ¹H nuclear magneticresonance spectroscopy. The HPLC method and retention time, along withmass spectral data are provided in Table 2 below. HPLC methods used areas follows: Method A conditions were Waters C-18 column, 4.6×150 mm, 3.5micron, 23° C., 1.0 mL/min, 1 min 25% MeCN in H₂O (0.1% TFA), 10 mingradient of 25%-95% MeCN in H₂O (0.1% TFA), 95% MeCN in H₂O (0.1% TFA)for 5 min, and then equilibration to 25% MeCN in H₂O (0.1% TFA) over 2.0min; Method B conditions were Agilent Zorbax C-18 column, 4.6×50 mm, 1.8micron, 23° C., 1.0 mL/min, 1 min 25% MeCN in H₂O (0.1% TFA), 5 mingradient of 25%-95% MeCN in H₂O (0.1% TFA), 1 min at 95% MeCN in H₂O(0.1% TFA), and then equilibration to 25% MeCN in H₂O (0.1% TFA) over1.0 min; Method C conditions were Phenomenex Kinetex C18 (3.0 mm×50 mm),2.6 micron, 58° C., 1.5 mL/min, 4 min gradient 5% MeCN (0.1% TFA) in H₂O(0.1% TFA) to 100% MeCN (0.1% TFA), 100% MeCN (0.1% TFA) for 0.5 min,and then equilibration to 5% MeCN (0.1% TFA) in H₂O (0.1% TFA) over 1.5min; and Method D conditions were Phenomenex Kinetex C18 (3.0 mm×50 mm),2.6 micron, 40° C., 1.5 mL/min, 4 min gradient 5% MeCN (0.1% TFA) in H₂O(0.1% TFA) to 100% MeCN (0.1% TFA), 100% MeCN (0.1% TFA) for 0.5 min,and then equilibration to 5% MeCN (0.1% TFA) in H₂O (0.1% TFA) over 1.5min. The phrase “MeCN (0.1% TFA)” is art-recognized and refers toacetonitrile containing 0.1% wt/wt trifluoroacetic acid. The symbol “NA”indicates that data was not available.

¹H nuclear magnetic resonance data for exemplary compounds is providedin Table 3.

TABLE 2 HPLC Calculated MS Retention Compound MW (EI+) HPLC Time No.Chemical Structure (g/mol) m/z Method (min) A-1

468.35 469 B  7.09 A-2

452.79 452.06 D  2.94 A-3

436.33 437 B  6.85 A-4

420.77 420.06 D  3.02 A-5

452.79 452.82, 454.78 A 12.40 A-6

440.42 440.91 A 10.20 A-7

434.80 434.81 D  2.80 A-8

419.24 419.25 D  2.88 A-9

420.77 472.79, 474.81 A 15.20 A-10

434.80 434.81, 436.83 A  7.47 A-11

426.39 427.00 B  5.99 A-12

458.41 459.00 B  6.30 A-13

424.44 424.97 A  9.76 A-14

456.46 456.95 A 10.30 A-15

434.80 434.07 D  2.79 A-16

434.80 434.07 D  2.32 A-17

468.35 468.10 D  3.01 A-18

452.79 452.06 D  3.09 A-19

401.25 400.04 D  2.74 A-20

434.80 434.07 D  2.90 A-21

419.24 418.03 D  3.04 A-22

452.79 452.06 D  3.19 A-23

437.23 436.03 D  3.19 A-24

402.78 402.06 D  2.80 A-25

401.25 400.04 D  2.71 A-26

440.42 441.00 B  6.71 A-27

434.8 434.8 B  6.17 A-28

466.8 466.8 B  6.48 A-29

420.4 420.96 B  5.62 A-30

442.4 443 B  5.68 A-31

410.4 411 B  5.33 A-32

482.4 482.9 B  6.78 A-33

482.4 482.9 B  6.56 A-34

482.4 482.9 B  6.62 A-35

450.4 450.9 B  6.34 A-36

450.4 NA A 10.96 A-37

468.4 468.9 B  6.63 A-38

468.4 468.9 B  6.47 A-39

420.4 421 B  5.88 A-40

378.35 378.92 A  7.84 A-41

380.36 380.93 A 11.24 A-42

420.4 421 B  5.75 A-43

420.4 421 B  5.69 A-44

364.8 364.91, 386.82 (+Na) B 11.8 A-45

348.35 348.9 B 10.55 A-46

378.35 378.92 A  6.61 A-47

392.37 392.9 A 10.76 A-48

364.32 365 B  4.29 A-49

332.3 333 B  3.81 A-50

348.76 349 B  4.17 A-51

434.34 434.86 A 12 A-52

406.28 406.86 A 10.92 A-53

419.4 419.95 B  5.67 A-54

387.3 387.96 B  5.29 A-55

403.8 403.9 B  5.79 A-56

372.73 372.84, 374.79 A 10.56 A-57

400.78 400.84, 402.80 A 11.91 A-58

358.82 358.90, 360.88 A  9.43 A-59

418.41 418.92 A  9.51 A-60

434.8 434.79 B 10.57 A-61

406.4 406.9 A 11.65 A-62

372.85 372.89, 374.85 A 10.42 A-63

474.4 474.88 A  9.24,  9.85 A-64

440.85 440.83, 442.82 A  8.3,  8.74 A-65

508.9 NA B 12.43 A-66

480.84 481 B  7.58 A-67

462.86 463 C  2.93 A-68

462.86 463 C  2.83 A-69

462.86 463 C  2.81 A-70

418.35 419 C  2.55 A-71

416.82 417 C  2.57 A-72

416.82 417 C  2.59 A-73

450.37 451 C  2.73 A-74

450.37 451 C  2.68 A-75

436.34 437 C  2.69 A-76

415.28 415 C  2.5 A-77

448.84 449 C  2.67 A-78

448.84 449 C  2.61 A-79

448.84 449 C  2.62 A-80

416.82 417 C  2.47 A-81

433.27 433 C  2.57 A-82

433.27 433 C  2.57 A-83

433.27 433 C  2.6 A-84

433.27 433 C  2.61 A-85

432.38 433 C  2.31 A-86

450.37 451 C  2.46 A-87

415.28 415 C  2.23 A-88

415.28 415 C  2.27 A-89

392.38 393 C  2.06 A-90

394.4 395 C  2.54 A-91

434.47 435 C  2.41 A-92

408.38 409 C  1.86 A-93

408.38 409 C  1.94 A-94

396.37 397 C  1.92 A-95

360.37 361 C  1.89 A-96

362.38 363 C  2.88 A-97

402.45 403 C  2.28 A-98

376.37 377 C  1.65 A-99

376.37 377 C  1.75 A-100

364.35 365 C  1.73 A-101

358.83 359 C  1.94 A-102

360.85 361 C  2.29 A-103

400.91 401 C  2.3 A-104

374.83 375 C  1.65 A-105

374.83 375 C  1.74 A-106

362.82 363 C  1.76 A-107

334.33 335 C  1.7 A-108

346.34 347 C  1.78 A-109

332.79 335 C  1.75 A-110

435.4 435.88 B  6.88 A-111

507.4 507.93 B  7.62 A-112

505.9 505.85 B  7.5 A-113

513.4 513.73 B  6.62 A-114

559.5 559.86 B  6.8 A-115

573.5 573.78 B  6.98 A-116

627.5 627.72 B  7.06 A-117

585.5 585.86 B  7.04 A-118

467.4 468 C  2.83 A-119

482.4 483 C  3.47 A-120

531.3 531 C  3.65 A-121

528.4 527.68 B  6.95 A-122

483.4 483.85 B  6.21 A-123

529.9 529.76 B  7.05 A-124

491.9 491.82 B  6.9 A-125

517.8 517.68 B  6.48 A-126

503.9 503.84 B  6.96 A-127

530 529.89 B  6.79 A-128

493.5 493.91 B  6.74 A-129

525.5 525.9 B  6.98 A-130

510.4 511.8 B  7.25 A-131

452 453.88 B  5.86 A-132

524.4 525.78 B  6.95 A-133

500 499.93 B  6.27 A-134

505.9 505.92 B  6.29 A-135

564.3 563.68 B  7.27 A-136

578.4 577.73 B  7.09 A-137

573.4 572.75 B  6.94 A-138

597.5 596.75 B  7.15 A-139

444 444.01 B  6.28 A-140

460.3 459.81 B  7.46 A-141

525 524.94 NA NA A-142

583.4 582.76 B  7.35 A-143

401.9 401.87 B  5.03 A-144

501 500.94 B  5.8 A-145

504 503.96 B  5.17 A-146

591.4 590.83 B  7.28 A-147

559.4 558.83 B  7.2 A-148

573.4 572.81 B  6.91 A-149

595.5 595.99 B  6.82 A-150

541.5 542.01 B  6.71 A-151

552.5 552.97 B  6.52

TABLE 3 Compound No. NMR Solvent ¹H NMR Resonance Data (δ) A-1 DMSO-d⁶13.00 (s, 1H), 12.31 (s, 1H), 11.40 (s, 1H), 8.25 (s, 1H), 8.18 (d, J =8.0 Hz, 2 H), 7.94 (d, J = 8.3 Hz, 1 H), 7.75 (d, J = 7.9 Hz, 2H), 7.75-7.57 (m, 3H), 6.67 (d, J = 7.9 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H) A-5DMSO-d⁶ 12.93 (s, 1H), 12.15 (s, 1H), 11.33 (s, 1H), 8.16 (d, 2H), 7.98(m, 1H), 7.8-7.7 (m, 4H), 7.50 (m, 1H), 6.65 (d, 1H), 6.43 (d, 1H) A-6DMSO-d⁶ 12.96 (s, 1H), 12.13 (s, 1H), 11.17 (s, 1H), 8.06 (d, 2H), 7.72(m, 3H), 7.63 (d, 1H), 7.25 (m, 2H), 7.07 (m, 1H), 6.66 (d, 1H), 6.42(d, 1H), 2.05 (m, 1H), 0.87 (m, 2H), 0.64 (m, 2H) A-14 DMSO-d⁶ 12.95 (brs, 1H), 11.97 (s, 1H), 11.10 (s, 1H), 7.94 (m, 2H), 7.74- 7.66 (m, 3H),7.5 (m, 1H), 7.32 (m, 3H), 6.67 (m, 1H), 6.41 (m, 1H), 1.34 (s, 1H) A-26DMSO-d⁶ 12.96 (s, 1H), 12.05 (s, 1H), 11.33 (s, 1H), 8.19 (d, J = 8.4Hz, 2 H), 7.78 (d, J = 7.9 Hz), 7.72 (t, J = 8.1 Hz, 1 H), 7.42 (d, J =7.5, 1H), 7.41 (s, 1H), 7.31 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 7.5 Hz,1H), 6.63 (d, J = 7.9 Hz, 1H), 6.42 (d, J = 7.9 Hz, 1H), 1.60 (m, 1H),0.96 (dd, J = 8.3, 2.1 Hz, 2H), 0.70 (dd, J = 4.8, 2.1, 2H) A-27 DMSO-d⁶12.75 (s, 1H), 11.04 (s, 1H), 10.88 (t, 1H), 7.92 (m, 2H), 7.67 (m, 2H),7.44 (m, 3H), 6.55 (d, 1H), 6.36 (d, 1H), 4.77 (d, 2H) A-28 DMSO-d⁶12.80 (s, 1H), 11.05 (s, 1H), 10.92 (t, 1H), 8.24 (d, 2H), 7.75 (d, 2H),7.70 (m, 2H), 7.42 (m, 2H), 6.58 (d, 1H), 6.38 (d, 1H), 4.80 (d, 2H)A-29 DMSO-d⁶ 12.71 (s, 1H), 11.10 (bs, 1H), 10.74 (bs, 1H), 8.27 (d,2H), 7.81 (d, 2H), 7.67 (t, 1H), 6.56 (d, 1H), 6.42 (d, 1H), 4.42 (bs,1H), 1.83 (m, 2H), 1.68 (m, 6H), 1.34 (m, 2H), 0.95 (d, 3H) A-32 DMSO-d⁶12.84 (s, 1H), 11.02 (d, 2H), 8.08 (d, 2H), 7.77 (d, 1H), 7.73-7.63 (m,4H), 7.56 (d, 1H), 7.46 (t, 1H), 6.62 (d, 1H), 6.39 (d, 1H), 5.02 (d,2H) A-33 DMSO-d⁶ 12.78 (s, 1H), 11.06 (s, 1H), 10.98 (t, 1H), 8.20 (d,2H), 7.81 (s, 1H), 7.74-7.66 (m, 4H), 7.59 (m, 2H), 6.57 (d, 1H), 6.37(d, 1H), 4.88 (d, 2H) A-34 DMSO-d⁶ 12.82 (s, 1H), 11.06 (s, 1H), 10.98(t, 1H), 8.20 (d, 2H), 7.76-7.62 (m, 7H), 6.58 (d, 1H), 6.37 (d, 1H),4.90 (d, 2H) A-35 DMSO-d⁶ 12.76 (s, 1H), 11.05 (s, 1H), 10.96 (t, 1H),7.85 (m, 2H), 7.72-7.60 (m, 5H), 7.43 (q, 1H), 6.56 (d, 1H), 6.36 (d,1H), 4.88 (d, 2H) A-36 DMSO-d⁶ 12.82 (s, 1H), 11.05 (s, 1H), 10.91 (t,1H), 8.25 (d, 2H), 7.76 (d, 2H), 7.68 (t, 1H), 7.49-7.36 (m, 2H), 7.27(m, 1H), 6.57 (d, 1H), 6.36 (d, 1H), 4.80 (d, 2H) A-37 DMSO-d⁶ 12.81 (s,1H), 11.04 (s, 1H), 10.91 (t, 1H), 8.23 (d, 2H), 7.76 (d, 2H), 7.68 (t,1H), 7.36 (t, 2H), 6.58 (d, 1H), 6.37 (d, 1H), 4.80 (d, 2H) A-38 DMSO-d⁶12.76 (s, 1H), 11.14 (s, 1H), 10.81 (t, 1H), 8.27 (d, 2H), 7.79 (d, 2H),7.66 (t, 1H), 7.19 (t, 2H), 6.53 (d, 1H), 6.37 (d, 1H), 4.82 (d, 2H)A-39 DMSO-d⁶ 12.79 (s, 1H), 11.11 (bs, 1H), 10.16 (d, 1H), 8.28 (d, 2H),7.80 (d, 2H), 7.65 (t, 1H), 6.50 (d, 1H), 6.36 (d, 1H), 4.37 (m, 1H),1.99 (m, 2H), 1.80-1.55 (m, 10H) A-43 DMSO-d⁶ 12.85 (s, 1H), 11.13 (s,1H), 10.09 (d, 1H), 8.26 (d, 2H), 7.80 (d, 2H), 7.65 (t, 1H), 6.50 (d,1H), 6.35 (d, 1H), 3.94 (q, 1H), 1.99 (bd, 1H), 1.81-1.67 (m, 4H),1.50-1.11 (m, 5H), 0.92 (d, 3H) A-44 DMSO-d⁶ 12.82 (s, 1H), 11.01 (s,1H), 10.21 (s, 1H), 7.91 (dd, 1H), 7.91 (s, 1H), 7.72-7.62 (m, 2H), 6.48(d, 1H), 6.38 (d, 1H), 1.54 (s, 9H) A-45 DMSO-d⁶ 8.50-8.40 (m, 1H), 7.75(d, 0.5H), 7.60-7.50 (m, 6H), 7.50-7.40 (m, 0.5H), 1.50 (s, 9H) A-53DMSO-d⁶ 12.83 (s, 1H), 11.10 (s, 1H), 10.84 (t, 1H), 8.28 (d, 2H), 7.78(d, 2H), 7.68 (t, 1H), 6.57 (d, 1H), 6.36 (d, 1H), 6.22 (s, 1H), 4.80(d, 2H), 2.32 (s, 3H) A-60 DMSO-d⁶ 12.86 (1H, s), 11.0 (s, 1H), 10.83(s, 1H), 8.05 (d, 2H), 7.67 (dd, 1H), 7.45 (d, 2H), 7.35 (dd, 2H), 6.66(d, 1H), 6.36 (d, 1H), 4.78 (d, 2H) A-65 CDCl₃ 13.38 (s, 1H), 10.22 (s,1H), 8.29 (d, 2H), 7.77 (dd, 1H), 7.66 (d, 2H), 7.49 (dd, 1H), 7.3-7.25(m, 1H), 7.13 (dd, 1H), 6.84 (d, 1H), 6.72 (d, 1H), 4.81 (d, 2H), 2.27(s, 3H) A-110 DMSO-d6 12.88 (s, 1H), 12.79 (s, 1H), 8.43 (s, 1H),7.93-7.85 (m, 3H), 7.63 (t, 1H, J = 8 Hz), 7.55-7.43 (m, 3H), 6.64 (s,2H), 6.22 (t, 2H, J = 8.4 Hz) A-111 DMSO-d6 13.09 (s, 1H), 12.28 (s,1H), 10.64 (s, 1H), 8.13 (s, 1H), 7.91 (d, 1H, J = 8.4 Hz), 7.85-7.79(m, 3H), 7.66 (t, 1H, J = 8 Hz), 7.59 (d, 1H, J = 8 Hz), 7.45 (q, 1H, J= 8.4 Hz), 7.21 (d, 1H, J = 8 Hz), 6.81 (d, 1H, J = 8 Hz), 4.15 (q, 2H,J = 6.8 Hz), 1.24 (t, 3H, J = 6.8 Hz) A-112 DMSO-d6 12.84 (s, 1H), 10.89(t, 1H, J = 6 Hz), 10.48 (s, 1H), 7.91-7.84 (m, 2H), 7.77 (t, 1H, J = 8Hz), 7.47-7.36 (m, 4H), 7.25 (dd, 1H, J = 8.4, 1.6 Hz), 6.73 (d, 1H, J =8 Hz), 4.81 (d, 2H, J = 7.2 Hz), 4.15 (q, 2H, J = 7.2 Hz), 4.06 (q, 1H,J = 5.2 Hz), 3.14 (d, 2H, J = 5.2 Hz), 1.24 (t, 3H, J = 6.8 Hz) A-113DMSO-d6 3.13 (s, 1H), 11.69 (s, 1H), 11.10 (s, 1H), 8.14 (s, 1H),7.86-7.78 (m, 4H), 7.66 (t, 1H, J = 8 Hz), 7.59 (d, 1H, J = 8 Hz), 7.47(m, 1H), 6.87 (d, 1H, J = 8 Hz), 6.72 (d, 1H, J = 8 Hz), 3.25 (s, 3H)A-114 DMSO-d6 12.92 (s, 1H), 10.96 (s, 1H), 10.81 (t, 1H, J = 6 Hz),8.05 (d, 2H, J = 8 Hz), 7.80 (t, 1H, J = 8 Hz), 7.73 (d, 1H, J = 7.6Hz), 7.67 (d, 2H, J = 8 Hz), 7.62 (t, 1H, J = 7.6 Hz), 7.52 (d, 1H, J =6.8 Hz), 7.43 (t, 1H, J = 7.6 Hz), 6.83 (d, 1H, J = 8 Hz), 6.69 (d, 1H,J = 8 Hz), 4.97 (d, 2H, J = 6Hz), 3.14 (s, 3H) A-115 DMSO-d6 12.91 (s,1H), 10.91 (s, 1H), 10.84 (t, 1H, J = 5.2 Hz), 8.06 (d, 2H, J = 8 Hz),7.82-7.73 (m, 2 H), 7.68 (d, 2 H, J = 7.6 Hz), 7.60 (t, 1H, J = 7.6 Hz),7.52 (d, 1H, J = 7.6 Hz), 7.43 (t, 1H, J = 7.6 Hz), 6.83 (d, 1H, J = 8Hz), 6.71 (dd, 1H, J = 8, 2 Hz), 4.97 (d, 2H, J = 5.2 Hz), 3.25 (q, 2H,J = 8 Hz), 1.10 (t, 3H, J = 8 Hz) A-116 DMSO-d6 12.93 (s, 1H), 11.58 (s,1H), 10.72 (t, 1H, J = 5.2 Hz), 8.04 (d, 2H, J = 7.6 Hz), 7.82 (t, 1H, J= 8 Hz), 7.73 (d, 1H, J = 8 Hz), 7.67 (d, 2H, J = 8 Hz), 7.58-7.52 (m,2H), 7.42 (t, 1H, J = 7.2 Hz), 6.89 (d, 1H, J = 8 Hz), 6.73 (d, 1H, J =8 Hz), 4.96 (d, 2H, J = 5.2 Hz), 4.75 (q, 2H, J = 9.6 Hz) A-117 DMSO-d612.92 (s, 1H), 10.91 (s, 2H), 8.05 (d, 2H, J = 7.6 Hz), 7.79 (td, 1H, J= 8, 2.4 Hz), 7.73 (d, 1H, J = 8 Hz), 7.67 (d, 2H, J = 8 Hz), 7.60 (t,1H, J = 7.6 Hz), 7.53 (d, 1H, J = 7.6 Hz), 7.42 (t, 1H, J = 7.6 Hz),6.83 (d, 1H, J = 8 Hz), 6.73 (d, 1H, J = 7.6 Hz), 4.97 (d, 2 H, J = 5.6Hz), 2.92 (m, 1 H), 0.96-0.91 (m, 4H) A-121 DMSO-d6 12.79 (s, 1H), 11.01(s, 1H), 10.76 (t, 1H, J = 6 Hz), 7.92-7.89 (m, 2H), 7.77 (t, 1H, J = 8Hz), 7.66-7.61 (m, 2H), 7.40-7.34 (m, 2H), 6.77 (d, 1H, J = 8 Hz), 6.64(d, 1H, J = 8 Hz), 4.74 (d, 2H, J = 6 Hz), 3.17 (s, 3H) A-122 DMSO-d612.92 (s, 1H), 11.06 (s, 1H), 10.57 (m, 1H), 8.32 (d, 2H, J = 8 Hz),7.84-7.79 (m, 3H), 6.85 (d, 1H, J = 8 Hz), 6.68 (d, 1H, J = 7.6 Hz),4.41 (m, 2H), 3.23 (s, 3H) A-123 DMSO-d6 13.10 (s, 1H), 11.63 (s, 1H),11.09 (s, 1H), 8.17 (d, 2H, J = 7.6 Hz), 7.86-7.80 (m, 3H), 7.68 (s,1H), 7.53 (dd, 1H, J = 10.4, 1.6 Hz), 7.31 (d, 1H, J = 8.8 Hz), 6.89 (d,1H, J = 8 Hz), 6.74 (d, 1H, J = 8 Hz), 3.24 (s, 3H) A-124 DMSO-d6 13.27(s, 1H), 11.34 (s, 1H), 8.96 (s, 1H), 8.09 (dd, 2H, J = 6.8, 2 Hz), 7.78(t, 1H, J = 8 Hz), 7.53 (dd, 2H, J = 6.8, 2 Hz), 6.76 (d, 2H, J = 8 Hz),4.74 (q, 2H, J = 9.6 Hz), 1.55 (s, 9H) A-125 DMSO-d6 12.99 (s, 1H),11.70 (bs, 1H), 10.37 (t, 1H, J = 6 Hz), 8.55 (m, 1H), 8.14 (d, 2H, J =7.2 Hz), 7.83-7.75 (m, 1H), 7.52 (d, 2H, J = 6.8 Hz), 7.37 (m, 1H), 6.87(d, 1H, J = 7.6 Hz), 6.70 (d, 1H, J = 8 Hz), 4.78 (q, 2H, J = 9.6 Hz),4.38 (quint, 2H, J = 8.8 Hz) A-126 DMSO-d6 13.16 (s, 1H), 11.53 (s, 1H),9.53 (s, 1H), 8.08 (d, 2H, J = 8.4 Hz), 7.76 (t, 1H, J = 8 Hz), 7.50 (d,2H, J = 8 Hz), 6.74 (d, 1H, J = 8 Hz), 6.64 (d, 1H, J = 7.6 Hz), 4.75(q, 2H, J = 10 Hz), 2.40 (q, 2H, J = 12 Hz), 2.17 (bt, 2H, J = 9.6 Hz),1.85-1.77 (m, 2H), 1.63 (s, 3H) A-127 DMSO-d6 12.91 (s, 1H), 11.40 (s,1H), 9.58 (d, 1H, J = 8.8 Hz), 7.76 (d, 2H, J = 7.6 Hz), 7.54 (t, 1H, J= 7.6 Hz), 7.22 (d, 2H, J = 7.6 Hz), 6.52 (d, 1H, J = 7.6 Hz), 6.40 (d,1H, J = 8 Hz), 4.51 (q, 2H, J = 9.6 Hz), 2.79 (m, 1H), 1.04 (m, 2H),0.31-0.01 (m, 8H) A-128 DMSO-d6 13.13 (s, 1H), 11.35 (bs, 1H), 9.00 (s,1H), 7.96 (m, 2H), 7.78 (t, 1H, J = 8 Hz), 7.55 (q, 1H, J = 8.4 Hz),6.76 (d, 2H, J = 8 Hz), 4.74 (q, 2H, J = 10Hz), 1.55 (s, 9H) A-129DMSO-d6 13.18 (s, 1H), 11.36 (s, 1H), 9.03 (s, 1H), 8.27 (d, 2H, J = 8Hz), 7.84 (d, 2H, J = 8.4), 7.78 (t, 1H, J = 8 Hz), 6.77 (t, 2H, J = 6Hz), 4.73 (q, 2H, J = 10 Hz), 1.56 (s, 9H) A-130 DMSO-d6 13.14 (s, 1H),11.56 (s, 1H), 11.04 (s, 1H), 7.99 (d, 2H, J = 8.4 Hz), 7.81 (t, 1H, J =8 Hz), 7.68 (s, 1H), 7.55-7.49 (m, 3H), 7.29 (dt, 1H, J = 8.4, 2 Hz),6.85 (d, 1H, J = 8 Hz), 6.72 (d, 1H, J = 8 Hz), 3.35 (q, 2H, J = 7.2Hz), 1.16 (t, 3H, J = 7.2 Hz) A-132 DMSO-d6 12.96 (s, 1H), 10.93 (s,1H), 10.64 (s, 1H), 8.02 (d, 2H, J = 8.4 Hz), 7.76 (t, 1H, J = 8 Hz),7.46-7.40 (m, 4H), 7.23 (dd, 1H, J = 8.4, 1.6 Hz), 6.76 (d, 1H, J = 7.6Hz), 6.66 (d, 1H, J = 8 Hz), 4.78 (d, 2H, J = 6 Hz), 3.25 (q, 2H, J =7.2 Hz), 1.14 (t, 3H, J = 7.2 Hz) A-135 DMSO-d6 13.16 (s, 1H), 11.68(bs, 1H), 11.41 (s, 1H), 8.00 (d, 2H, J = 8.8 Hz), 7.84 (t, 1H, J = 8Hz), 7.64 (s, 1H), 7.51-7.46 (m, 3H), 7.28 (d, 1H, J = 8.8 Hz), 6.92 (d,1H, J = 8 Hz), 6.76 (d, 1H, J = 8.4 Hz), 4.84 (q, 2H, J = 9.6 Hz) A-136DMSO-d6 12.99 (s, 1H), 11.60 (s, 1H), 10.51 (s, 1H), 8.01 (d, 2H, J =8.4 Hz), 7.79 (t, 1H, J = 8 Hz), 7.46-7.40 (m, 4H), 7.21 (dd, 1H, J =8.4, 2 Hz), 6.83 (d, 1H, J = 8 Hz), 6.70 (d, 1H, J = 8 Hz), 4.76 (m, 4H)A-137 DMSO-d6 12.87 (s, 1H), 11.67 (s, 1H), 10.64 (t, 1H, J = 6 Hz),8.64 (d, 1H, J = 4.8 Hz), 8.05-7.98 (m, 3H), 7.92 (d, 1H, J = 8 Hz),7.71-7.62 (m, 2H), 7.45-7.36 (m, 4H), 7.25 (dd, 1H, J = 8, 1.6 Hz), 6.71(t, 2H, J = 8.4 Hz), 4.84 (d, 2H, J = 6 Hz) A-138 DMSO-d6 12.90 (s, 1H),12.01 (s, 1H), 10.49 (t, 1H, J = 6 Hz), 8.06 (dd, 1H, J = 7.2, 1.6 Hz),7.98 (m, 3H), 7.87-7.72 (m, 3H), 7.45-7.41 (m, 3H), 7.32 (t, 1H, J = 8Hz), 7.21 (dd, 1H, J = 8.4, 2 Hz), 6.78 (d, 1H, J = 8 Hz), 6.72 (d, 1H,J = 8 Hz), 4.80 (d, 2H, J = 6 Hz) A-139 DMSO-d6 13.02 (s, 1H), 10.58 (t,1H, J = 6.4 Hz), 9.60 (s, 1H), 8.11 (d, 2H, J = 8.4 Hz), 7.78 (t, 1H, J= 8 Hz), 7.49 (d, 2H, J = 8.4 Hz), 7.40 (d, 1H, J = 7.6 Hz), 6.79 (d,1H, J = 8 Hz), 3.53 (d, 2H, J = 6.4 Hz), 1.21 (s, 9H), 0.93 (s, 9H)A-140 DMSO-d6 13.11 (s, 1H), 12.24 (s, 1H), 10.68 (s, 1H), 8.00 (d, 2H,J = 8.8 Hz), 7.82 (m, 2H), 7.67 (d, 1H, J = 10.8 Hz), 7.49 (d, 2H, J =8.8 Hz), 7.29 (m, 2H), 6.85 (d, 1H, J = 8 Hz), 2.13 (s, 3H) A-142DMSO-d6 13.00 (s, 1H), 12.08 (bs, 1H), 11.53 (s, 1H), 8.05-7.96 (m, 4H),7.83-7.77 (m, 3H), 7.59 (s, 1H), 7.49 (m, 2H), 7.30 (d, 1H, J = 8.4 Hz),7.21 (t, 1H, J = 7.6 Hz), 7.13 (m, 2H), 6.89 (d, 1H, J = 8 Hz), 6.79 (d,1H, J = 7.6 Hz) A-144 DMSO-d6 13.03 (s, 1H), 11.57 (s, 1H), 10.08 (s,1H), 8.96 (d, 1H, J = 2.4 Hz), 8.77 (dd, 1H, J = 4.8, 1.2 Hz), 8.17 (dt,1H, J = 8, 1.6 Hz), 8.11 (d, 2H, J = 8.8 Hz), 7.70 (t, 1H, J = 8 Hz),7.60 (dd, 1H, J = 8, 4.8 Hz), 7.49 (d, 2H, J = 8.8 Hz), 6.70 (m, 2H),3.52 (d, 2H, J = 6.4 Hz), 0.91 (s, 9H) A-145 DMSO-d6 13.04 (s, 1H),11.19 (s, 1H), 10.21 (t, 1H, J = 6.4 Hz), 8.10 (d, 2H, J = 8.4 Hz), 7.84(s, 1H), 7.72 (s, 1H), 7.66 (t, 1H, J = 8.4 Hz), 7.49 (d, 2H, J = 8.4Hz), 6.72 (d, 1H, J = 8 Hz), 6.64 (d, 1H, J = 8 Hz), 3.64 (s, 3H), 3.51(d, 2H, J = 6.4 Hz), 0.92 (s, 9H) A-146 DMSO-d6 12.96 (s, 1H), 11.63 (s,1H), 10.54 (t, 1H, J = 5.6 Hz), 8.01 (d, 2H, J = 8.4 Hz), 7.77 (t, 1H, J= 8 Hz), 7.46-7.37 (m, 4H), 7.25 (dd, 1H, J = 8, 1.6 Hz), 6.83 (d, 1H, J= 8 Hz), 6.74 (d, 1H, J = 8 Hz), 4.81 (d, 2H, J = 6.4 Hz), 2.58 (s, 3H),2.28 (s, 3H) A-147 DMSO-d6 13.05 (s, 1H), 11.77 (bs, 1H), 11.58 (s, 1H),8.94 (d, 1H, J = 2 Hz), 8.77 (dd, 1H, J = 4.4, 1.2 Hz), 8.16 (d, 1H, J =8 Hz), 7.97 (d, 2H, J = 8.4 Hz), 7.78 (t, 1H, J = 8 Hz), 7.66 (s, 1H),7.61-7.48 (m, 4H), 7.32 (d, 1H, J = 8.8 Hz), 6.86 (d, 1H, J = 8.4 Hz),6.77 (d, 1H, J = 8 Hz) A-149 DMSO-d6 12.88 (s, 1H), 11.60 (s, 1H), 10.72(t, 1H, J = 5.6 Hz), 7.83 (t, 1H, J = 8 Hz), 7.76-7.69 (m, 3H),7.61-7.52 (m, 2H), 7.45-7.36 (m, 2H), 6.88 (d, 1H, J = 8 Hz), 6.74 (d,1H, J = 8 Hz), 4.93 (d, 2H, J = 5.6 Hz), 4.78 (q, 2H, J = 9.6 Hz) A-150DMSO-d6 12.82 (s, 1H), 10.93 (s, 1H), 10.84 (t, 1H, J = 6 Hz), 7.82-7.71(m, 4H), 7.61 (m, 1H), 7.52 (d, 1H, J = 8 Hz), 7.46-7.36 (m, 2H), 6.82(d, 1H, J = 8 Hz), 6.71 (d, 1H, J = 8 Hz), 4.94 (d, 2H, J = 5.6 Hz),3.28 (m, 2H), 1.13 (m, 3H) A-151 DMSO-d6 12.88 (s, 1H), 11.85 (bs, 1H),10.68 (t, 1H, J = 5.6 Hz), 7.85 (t, 1H, J = 8 Hz), 7.77-7.69 (m, 3H),7.62-7.54 (m, 2H), 7.45-7.36 (m, 2H), 6.91 (d, 1H, J = 8 Hz), 6.76 (d,1H, J = 8 Hz), 5.17 (s, 2H), 4.93 (d, 2H, J = 5.6 Hz)

Example 6

The compounds listed in Table 4 were tested for activity againstF₁F₀-ATPase by measuring the ability of the compounds to inhibit ATPsynthesis. In addition, the compounds were assessed for cytotoxicity inRamos cells Inhibition of F₁F₀-ATPase activity in synthesizing ATP andcytotoxicity in Ramos cells were measured according to the proceduresdescribed in K. M. Johnson et al. Chemistry & Biology 2005, 12, 485-496.

TABLE 4 Compound No. ATP Syn IC₅₀ (μM) Ramos Cell EC₅₀ (μM) A-1 <10 <10A-2 <10 <10 A-3 <10 <10 A-4 <10 <10 A-5 <10 <10 A-6 <10 <10 A-7 <10 <10A-8 <10 <10 A-9 >10 >10 A-10 >10 >10 A-11 <10 <10 A-12 <10 <10 A-13 <10<10 A-14 <10 <10 A-15 <10 <10 A-16 <10 <10 A-17 <10 <10 A-18 <10 <10A-19 <10 <10 A-20 <10 <10 A-21 <10 <10 A-22 <10 <10 A-23 <10 <10 A-24<10 <10 A-25 <10 >10 A-26 <10 <10 A-27 <10 <10 A-28 <10 <10 A-29 <10 <10A-30 <10 <10 A-31 <10 <10 A-32 <10 <10 A-33 <10 <10 A-34 <10 <10 A-35<10 <10 A-36 <10 <10 A-37 <10 <10 A-38 <10 <10 A-39 <10 <10 A-40 <10 <10A-41 <10 <10 A-42 <10 <10 A-43 <10 >10 A-44 <10 <10 A-45 <10 <10 A-46<10 <10 A-47 <10 <10 A-48 <10 >10 A-49 >10 >10 A-50 <10 >10 A-51 <10 <10A-52 <10 <10 A-53 <10 <10 A-54 <10 >10 A-55 <10 >10 A-56 <10 <10 A-57<10 <10 A-58 <10 <10 A-59 <10 <10 A-60 <10 >10 A-61 <10 <10 A-62 <10 <10A-63 <10 <10 A-64 <10 <10 A-65 <10 <10 A-66 >10 >10 A-67 <10 <10 A-68<10 <10 A-69 <10 <10 A-70 <10 <10 A-71 <10 <10 A-72 <10 <10 A-73 <10 <10A-74 <10 <10 A-75 >10 >10 A-76 <10 <10 A-77 <10 <10 A-78 <10 <10 A-79<10 <10 A-80 <10 <10 A-81 <10 <10 A-82 <10 <10 A-83 <10 <10 A-84 <10 >10A-85 <10 <10 A-86 <10 <10 A-87 <10 <10 A-88 <10 <10 A-89 <10 <10 A-90<10 <10 A-91 <10 <10 A-92 <10 <10 A-93 <10 <10 A-94 <10 >10 A-95 <10 <10A-96 <10 <10 A-97 <10 <10 A-98 >10 >10 A-99 >10 >10 A-100 >10 >10 A-101<10 >10 A-102 <10 <10 A-103 <10 <10 A-104 >10 >10 A-105 >10 >10A-106 >10 >10 A-107 <10 >10 A-108 <10 >10 A-109 <10 >10 A-110 <10 >10A-111 >10 >10 A-112 <10 >10 A-113 <10 <10 A-114 <10 <10 A-115 <10 <10A-116 <10 <10 A-117 <10 <10 A-118 >10 >10 A-119 >10 >10 A-120 >10 >10A-121 <10 <10 A-122 <10 >10 A-123 <10 <10 A-124 <10 <10 A-125 <10 <10A-126 <10 <10 A-127 <10 <10 A-128 <10 <10 A-129 <10 <10 A-130 <10 <10A-131 <10 <10 A-132 <10 <10 A-133 <10 >10 A-134 <10 <10 A-135 <10 <10A-136 <10 <10 A-137 <10 >10 A-138 <10 <10 A-139 >10 >10 A-140 <10 >10A-141 <10 <10 A-142 <10 <10 A-143 <10 <10 A-144 <10 >10 A-145 >10 >10A-146 <10 <10 A-147 <10 <10 A-148 <10 <10 A-149 <10 <10 A-150 <10 >10A-151 <10 <10

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A compound represented by Formula I:

including all stereoisomers, geometric isomers, and tautomers; or apharmaceutically acceptable salt or solvate of any of the foregoing;wherein: A¹ is phenylene or a six-membered heteroarylene; A² is

X is halogen, haloalkyl, C₁-C₆alkoxy, —N(H)(R⁸), or —OP(O)(OR¹¹)₂; R¹represents independently for each occurrence halogen, alkyl, haloalkyl,hydroxyl, C₁-C₆alkoxy, or cyano; R² is hydrogen or alkyl; R³ is aryl,aralkyl, cycloalkyl, —(C(R⁶)₂)_(m)-cycloalkyl, heteroaryl,heteroaralkyl, heterocycloalkyl, —(C(R⁶)₂)_(m)-heterocycloalkyl, alkyl,haloalkyl, hydroxylalkyl, —(C(R⁶)₂)_(m)-alkoxyl, or —(C(R⁸)₂)_(m)—CN,wherein said aryl, aralkyl, cycloalkyl, heteroaryl, heteroaralkyl, andheterocycloalkyl are each optionally substituted with 1, 2, or 3substituents independently selected from the group consisting ofhalogen, haloalkyl, hydroxyl, alkyl, cycloalkyl, C₁-C₆alkoxy, and cyano;R⁴ is hydrogen, alkyl, or —C(O)R⁷; or R³ and R⁴ are taken together withthe nitrogen atom to which they are attached to form a 3 to 7 memberedheterocyclic ring optionally substituted with 1, 2, or 3 substituentsindependently selected from the group consisting of halogen, haloalkyl,hydroxyl, alkyl, cycloalkyl, and C₁-C₆alkoxy; R⁵ is hydrogen, halogen,alkyl, alkoxyl, or —C(O)R⁷; R⁶ represents independently for eachoccurrence hydrogen, alkyl, or cycloalkyl; R⁷ represents independentlyfor each occurrence alkyl or cycloalkyl; R⁸ is hydrogen or alkyl; R⁹ isalkyl, cycloalkyl, haloalkyl, —(C(R⁶)₂)_(m)-cycloalkyl,—(C(R⁶)₂)_(m)—CN, aryl, aralkyl, heteroaryl, or heteroaralkyl; R¹⁰ isalkyl, cycloalkyl, —(C(R⁶)₂)_(m)-cycloalkyl, haloalkyl, or C₁-C₆alkoxy;R¹¹ represents independently for each occurrence hydrogen or an alkalimetal; n is 0, 1, 2, or 3; and m is 1, 2, 3, 4, or
 5. 2. The compound ofclaim 1, wherein A¹ is phenylene.
 3. The compound of claim 1, wherein A²is


4. The compound of claim 2, wherein A² is


5. The compound of claim 1, wherein R¹ is halogen or haloalkyl.
 6. Thecompound of claim 4, wherein R¹ is chloro, fluoro, or trifluoromethyl.7. The compound of claim 4, wherein R² and R⁴ are hydrogen.
 8. Thecompound of claim 1, wherein R³ is aryl or aralkyl, each of which isoptionally substituted with 1, 2, or 3 substituents independentlyselected from the group consisting of halogen, haloalkyl, alkyl, andcycloalkyl.
 9. The compound of claim 1, wherein R³ is phenyl optionallysubstituted with 1, 2, or 3 substituents independently selected from thegroup consisting of halogen, haloalkyl, alkyl, and cycloalkyl.
 10. Thecompound of claim 1, wherein R³ is phenyl substituted with 1 or 2substituents independently selected from the group consisting of chloro,fluoro, and trifluoromethyl.
 11. The compound of claim 1, wherein R³ isbenzyl optionally substituted with 1, 2, or 3 substituents independentlyselected from the group consisting of halogen, haloalkyl, alkyl, andcycloalkyl.
 12. The compound of claim 1, wherein R³ is alkyl,hydroxyalkyl, cycloalkyl, or —(C(R⁶)₂)_(m)-alkoxyl, wherein saidcycloalkyl is optionally substituted with 1 or 2 substituentsindependently selected from the group consisting of halogen, haloalkyl,hydroxyl, and alkyl.
 13. The compound of claim 4, wherein R⁵ ishydrogen.
 14. The compound of claim 1, wherein n is 1 or
 2. 15. Thecompound of claim 1, wherein said compound is represented by FormulaI-A:

including all stereoisomers, geometric isomers, tautomers, or apharmaceutically acceptable salt or solvate of any of the foregoing;wherein: R¹ and R² each represent independently for each occurrencehydrogen, chloro, fluoro, or —CF₃; and R³ is aryl, aralkyl, heteroaryl,or heteroaralkyl, wherein said aryl, aralkyl, heteroaryl, andheteroaralkyl are each optionally substituted with 1, 2, or 3substituents independently selected from the group consisting ofhalogen, haloalkyl, hydroxyl, alkyl, C₁-C₆alkoxy, and cyano.
 16. Thecompound of claim 15, wherein R¹ and R² are independently chloro orfluoro.
 17. The compound of claim 16, wherein R³ is aryl or aralkyl,each of which is optionally substituted with 1, 2, or 3 substituentsindependently selected from the group consisting of halogen, haloalkyl,and alkyl.
 18. The compound of claim 16, wherein R³ is phenylsubstituted with 1 or 2 substituents independently selected from thegroup consisting of chloro, fluoro, and trifluoromethyl.
 19. Thecompound of 16, wherein R³ is benzyl substituted with 1 or 2substituents independently selected from the group consisting of chloro,fluoro, and trifluoromethyl.
 20. The compound of claim 1, wherein thecompound is one of compounds listed in Table 1 or 2, or apharmaceutically acceptable salt thereof.
 21. The compound of claim 1,wherein the compound is

or a pharmaceutically acceptable salt thereof.
 22. The compound of claim1, wherein the compound is


23. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 24. The pharmaceutical compositionof claim 23, wherein the compound is


25. A method of treating a disorder selected from the group consistingof an immune disorder, inflammatory disorder, cardiovascular disease,myeloma, lymphoma, and bacterial infection, comprising administering toa patient in need thereof a therapeutically effective amount of acompound of claim 1 in order to ameliorate a symptom of the disorder.26. A method of inhibiting a F₁F₀-ATPase, comprising exposing aF₁F₀-ATPase to a compound of claim 1 to inhibit said F₁F₀-ATPase.